Antagonist anti-il-7 receptor antibodies and methods

ABSTRACT

The present invention provides antagonizing antibodies that bind to interleukin-7 receptor (IL-7R). The invention further provides a method of obtaining such antibodies and antibody-encoding nucleic acids. The invention further relates to therapeutic methods for use of these antibodies and antigen-binding portions thereof for the treatment and/or prevention of type 2 diabetes and immunological disorders, including type 1 diabetes, multiple sclerosis, rheumatoid arthritis, graft-versus-host disease, and lupus.

This application is a divisional of U.S. patent application Ser. No.14/109,267, filed Dec. 17, 2013, which is a divisional of U.S. patentapplication Ser. No. 13/627,601, filed Sep. 26, 2012, now U.S. Pat. No.8,637,273, issued on Jan. 28, 2014, which is a divisional of U.S. patentapplication Ser. No. 13/033,491, filed Feb. 23, 2011, now U.S. Pat. No.8,298,535, issued on Oct. 30, 2012, which claims priority, under 35 USC§119(e), to U.S. Provisional Application Ser. No. 61/307,670, filed Feb.24, 2010, and U.S. Provisional Application Ser. No. 61/438,205, filedJan. 31, 2011, each of which is hereby incorporated by reference in itsentirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“PC33990D_SequenceListing_ST25.txt” created on Apr. 21, 2016 and havinga size of 41 KB. The sequence listing contained in this .txt file ispart of the specification and is herein incorporated by reference in itsentirety.

FIELD

The present invention relates to antibodies, e.g., full lengthantibodies or antigen-binding portions thereof, that antagonize theactivity of interleukin-7 receptor (IL-7R), including its interactionwith IL-7. The invention further relates to compositions comprising anIL-7R antagonist, such as an antagonist IL-7R antibody, and methods ofusing the IL-7R antagonist as a medicament. The IL-7R antagonist can beused in the prevention and/or treatment of type 2 diabetes,graft-versus-host disease (GVHD), and autoimmune disorders, includingtype 1 diabetes, multiple sclerosis, rheumatoid arthritis, and lupus.

BACKGROUND

The IL-7R complex is a heterodimeric receptor made up of the IL-7R alphachain (IL-7Rα) and the common gamma chain (γc) (Mazzucchelli et al., NatRev Immunol., 2007, 7:144-54). IL-7R is bound by interleukin-7 (IL-7), acytokine essential to the development and homeostatic maintenance of Tand B lymphocytes (Fry et al., J Immunol., 2005, 174:6571-6). Binding ofIL-7 to IL-7R activates multiple pathways that regulate lymphocytesurvival, glucose uptake, proliferation and differentiation.

IL-7R is expressed on both dendritic cells and monocytes and appears toact in multiple hematopoietic lineages (Reche P A, et al., J Immunol.,2001, 167:336-43). In dendritic cells, IL-7R plays an immunomodulatoryrole, whereas lymphocytes require IL-7R signaling for survival,proliferation and differentiation. Both the Jak-Stat and PI3K-Aktpathways are activated by the binding of IL-7 to IL-7R (Jian et al.,Cytokine Growth Factor Rev., 2005, 16:513-533). These pathways involvesignaling crosstalk, shared interaction domains, feedback loops,integrated gene regulation, mulitimerization and ligand competition.Some targets of IL-7 signaling, including Bcl2 and Pyk2, contribute tocellular survival. Other targets, such as PI3 kinase, src family kinases(Ick and fyn) and STAT5, contribute to cellular proliferation. Thetranscription factor STAT5 contributes to activation of multipledifferent downstream genes in B and T cells and may contribute to VDJrecombination through alteration of chromatin structure. The cellsurvival and cell proliferation signals induced by IL-7 combine toinduce normal T cell development. Details of the complex IL-7 signalingnetwork and its interaction with other signaling cascades in cells ofthe immune system have not yet been fully elucidated.

From the information available in the art, and prior to the presentinvention, it remained unclear whether the introduction of an antagonistIL-7R antibody into the blood circulation to selectively antagonizeIL-7R would be effective to treat type 2 diabetes, type 1 diabetes,GVHD, lupus and rheumatoid arthritis, and, if so, what properties of anIL-7R antibody are needed for such in vivo effectiveness.

SUMMARY

Antagonist antibodies that selectively interact with and inhibit IL-7Rfunction are provided. It is demonstrated for the first time thatcertain antagonist IL-7R antibodies are effective in vivo to treat type1 diabetes, type 2 diabetes, rheumatoid arthritis, GVHD and lupus.

In some embodiments, antagonist antibodies that selectively interactwith and inhibit IL-7R function are provided. In some embodiments, theantibody specifically binds to IL-7R and comprises an antigen bindingregion that competes with a monoclonal antibody selected from the groupconsisting of C1GM, C2M3, P3A9, P4B3, P2D2, P2E11, HAL403a and HAL403b,for binding to IL-7R. In some embodiments, the antibody comprises apolypeptide having the amino acid sequence shown in SEQ ID NO: 42 or SEQID NO: 43. In other embodiments, the antibody specifically binds toIL-7R and recognizes an epitope which overlaps an epitope of IL-7R thatis recognized by a monoclonal antibody selected from the groupconsisting of C1GM, C2M3, P3A9, P4B3, P2D2, P2E11, HAL403a and HAL403b.In some embodiments, the antibody the antibody binds to an epitopecomprising residues I82, K84, K100, T105, and Y192 of interleukin-7receptor alpha (IL-7Rα). In some embodiments, the epitope furthercomprises one or more additional residues selected from the groupconsisting of residues D190, H191, and K194 of human IL-7Rα.

In some embodiments, the IL-7R is human IL-7R.

In some embodiments, the antibody specifically binds to interleukin-7receptor alpha (IL-7Rα) and comprises a heavy chain variable region (VH)complementary determining region one (CDR1) having the amino acidsequence X₁X₂VMH, wherein X₁ is D or N; X₂ is S or Y (SEQ ID NO: 50), aVH CDR2 having the amino acid sequence X₁X₂X₃X₄X₅GX₆X₇TYYADSVKG, whereinX₁ is L or A; X₂ is V or I; X₃ is G or S; X₄ is W or G; X₅ is D or S; X₆is F, G or S; X₇ is F, A or S (SEQ ID NO: 51), and a VH CDR3 having theamino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein X₁ is Q or D; X₂ is G orI; X₃ is D or S; X₄ is Y or G; X₅ is M, V or G; X₆ is G or F; X₇ is N, Dor M; X₈ is N, Y or D (SEQ ID NO: 52), a light chain variable region(VL) CDR1 having the amino acid sequence TX₁SSGX₂IX₃SSYVQ wherein X₁ isR or G; X₂ is S or R; X₃ is D or A (SEQ ID NO: 53), a VL CDR2 having theamino acid sequence EDX₁QRPS wherein X₁ is D or N (SEQ ID NO: 54), and aVL CDR3 having the amino acid sequence X₁X₂YX₃X₄X₅X₆LX₇ wherein X₁ is Qor M; X₂ is S or Q; X₃ is D or A; X₄ is F or S; X₅ is H or S; X₆ is H orS; X₇ is V or W (SEQ ID NO: 55), wherein the antibody blocks STAT5phosphorylation in a STAT5 activation assay. In some embodiments, theframework region between VH CDR2 and VH CDR3 comprises the amino acidsequence alanine-arginine, wherein the arginine is adjacent to the firstamino acid residue of VH CDR3. In some embodiments, the framework regionbetween VH CDR2 and VH CDR3 comprises the amino acid sequencecysteine-alanine-arginine, wherein the arginine is adjacent to the firstamino acid residue of VH CDR3.

In some embodiments, the antibody specifically binds to IL-7Rα andcomprises a heavy chain variable region (VH) comprising the followingcomplementarity determining regions (CDRs): a VH CDR1 that is a VH CDR1in SEQ ID NO: 40; a VH CDR2 that is a VH CDR2 in SEQ ID NO: 40; and a VHCDR3 that is a VH CDR3 in SEQ ID NO: 40. In some embodiments, theantibody specifically binds to IL-7Rα and comprises a light chainvariable region (VL) comprising the following CDRs: a VL CDR1 that is aVL CDR1 in SEQ ID NO: 41; a VL CDR2 that is a VL CDR2 in SEQ ID NO: 41;and a VL CDR3 that is a VL CDR3 in SEQ ID NO: 41. In some embodiments,the antibody specifically binds to IL-7Rα and comprises: a heavy chainvariable region (VH) comprising the following complementaritydetermining regions (CDRs): a VH CDR1 that is a VH CDR1 in SEQ ID NO:40; a VH CDR2 that is a VH CDR2 in SEQ ID NO: 40; and a VH CDR3 that isa VH CDR3 in SEQ ID NO: 40; and a light chain variable region (VL)comprising the following CDRs: a VL CDR1 that is a VL CDR1 in SEQ ID NO:41; a VL CDR2 that is a VL CDR2 in SEQ ID NO: 41; and a VL CDR3 that isa VL CDR3 in SEQ ID NO: 41. In some embodiments, each CDR is defined inaccordance with the Kabat definition, the Chothia definition, thecombination of the Kabat definition and the Chothia definition, the AbMdefinition, or the contact definition of CDR.

In some embodiments, the antibody comprises a VH CDR1 having the aminoacid sequence X₁X₂VMH, wherein X₁ is D or N; X₂ is S or Y (SEQ ID NO:50), a VH CDR2 having the amino acid sequence GWDGFF (SEQ ID NO: 57),and a VH CDR3 having the amino acid sequence ARX₁X₂X₃X₄(SEQ ID NO: 58),a VL CDR1 having the amino acid sequence SGSIDSSY (SEQ ID NO: 59), a VLCDR2 having the amino acid sequence EDDQRPSGV (SEQ ID NO: 60), and a VLCDR3 having the amino acid sequence FHHL (SEQ ID NO: 61), wherein theantibody blocks STAT5 phosphorylation in a STAT5 activation assay.

In some embodiments, the antibody specifically binds to IL-7Rα andcomprises a heavy chain variable region (VH) complementary determiningregion one (CDR1) having the amino acid sequence DSVMH (SEQ ID NO: 19),GFTFDDS (SEQ ID NO: 46), or GFTFDDSVMH (SEQ ID NO: 47), a VH CDR2 havingthe amino acid sequence LVGWDGFFTYYADSVKG (SEQ ID NO: 23) or GWDGFF (SEQID NO: 48), and a VH CDR3 having the amino acid sequence QGDYMGNN (SEQID NO: 49), or a variant thereof having one or more conservative aminoacid substitutions in CDR1, CDR2, and/or CDR3.

In some embodiments, the antibody comprises a light chain variableregion (VL) CDR1 having the amino acid sequence TRSSGSIDSSYVQ (SEQ IDNO: 29), a VL CDR2 having the amino acid sequence EDDQRPS (SEQ ID NO:31), and/or VL CDR3 having the amino acid sequence QSYDFHHLV (SEQ ID NO:36), or a variant thereof having one or more conservative amino acidsubstitutions in CDR1, CDR2, and/or CDR3. In some embodiments, theantibody further comprises a VH CDR1 having the amino acid sequenceshown in SEQ ID NO: 19, 46 or 47, a VH CDR2 having the amino acidsequence shown in SEQ ID NO: 23, or 48, and a VH CDR3 having the aminoacid sequence shown in SEQ ID NO: 49, or a variant thereof having one ormore conservative amino acid substitutions in CDR1, CDR2, and/or CDR3.

In some embodiments, the antibody specifically binds to IL-7Rα andcomprises a heavy chain variable region (VH) complementary determiningregion one (CDR1) having the amino acid sequence shown in SEQ ID NO: 19,46 or 47, a VH CDR2 having the amino acid sequence shown in SEQ ID NO:23, or 48, and a VH CDR3 having the amino acid sequence shown in SEQ IDNO: 49, a light chain variable region (VL) CDR1 having the amino acidsequence shown in SEQ ID NO: 29, a VL CDR2 having the amino acidsequence shown in SEQ ID NO: 31, and a VL CDR3 having the amino acidsequence shown in SEQ ID NO: 36. In some embodiments, the VH regioncomprises the amino acid sequenceEVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSLVGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGTL VTVSS (SEQ IDNO: 40) and the VL region comprises the amino acid sequenceNFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVL (SEQ ID NO: 41). Insome embodiments, the antibody comprises a light chain having the aminoacid sequence NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 43) and a heavychain having the amino acid sequenceEVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSLVGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 42), with or without theC-terminal lysine of SEQ ID NO: 42.

In some embodiments, the antibody can be a human antibody, a humanizedantibody, or a chimeric antibody. In some embodiments, the antibody is amonoclonal antibody.

In some embodiments, the antibody comprises a constant region. In someembodiments, the antibody is of the human IgG1 or IgG2Δa subclass. Insome embodiments, the antibody comprises a glycosylated constant region.In some embodiments, the antibody comprises a constant region havingincreased binding affinity to a human Fc gamma receptor. In someembodiments, the antibody comprises an aglycosylated constant region.

In some embodiments, a pharmaceutical composition comprising an antibodythat selectively interacts with and inhibits IL-7R function is provided.

In some embodiments, a cell line that recombinantly produces an antibodythat selectively interacts with and inhibits IL-7R function is provided.

In some embodiments, a nucleic acid encoding an antibody thatselectively interacts with and inhibits IL-7R function is provided.

In some embodiments, methods of lowering blood glucose levels in anindividual are provided. In some embodiments, the method comprisesadministering a therapeutically effective amount of an antagonist IL-7Rantibody to an individual in need of such treatment, thereby loweringblood glucose levels.

In some embodiments, methods of improving glucose tolerance in anindividual are provided. In some embodiments, the method comprisesadministering a therapeutically effective amount of an antagonist IL-7Rantibody to an individual in need of such treatment, thereby improvingglucose tolerance.

In some embodiments, methods of preventing or treating type 1 diabetesin an individual are provided. In some embodiments, the method comprisesadministering a therapeutically effective amount of an antagonist IL-7Rantibody to an individual in need of such treatment, thereby preventingor treating one or more symptoms of type 1 diabetes.

In some embodiments, methods of preventing or treating type 2 diabetesin an individual are provided. In some embodiments, the method comprisesadministering a therapeutically effective amount of an IL-7R antagonistto an individual in need of such treatment, thereby preventing ortreating one or more symptoms of type 2 diabetes. In some embodiments,the IL-7R antagonist is an antagonist IL-7R antibody.

In some embodiments, methods of preventing or treating rheumatoidarthritis in an individual are provided. In some embodiments, the methodcomprises administering a therapeutically effective amount of anantagonist IL-7R antibody to an individual in need of such treatment,thereby preventing or treating one or more symptoms of rheumatoidarthritis.

In some embodiments, methods of preventing or treating graft-versus-hostdisease (GVHD) in an individual are provided. In some embodiments, themethod comprises administering a therapeutically effective amount of anantagonist IL-7R antibody to an individual in need of such treatment,thereby preventing or treating one or more symptoms of GVHD.

In some embodiments, the GVHD is chronic GVHD or acute GVHD.

In some embodiments, methods of preventing or treating lupus in anindividual are provided. In some embodiments, the method comprisesadministering a therapeutically effective amount of an antagonist IL-7Rantibody to an individual in need of such treatment, thereby preventingor treating one or more symptoms of lupus.

In some embodiments, the lupus is cutaneous lupus erythematosus,systemic lupus erythematosus, drug-induced erythematosus or neonatallupus.

In some embodiments, methods of preventing or treating multiplesclerosis in an individual are provided. In some embodiments, the methodcomprises administering a therapeutically effective amount of anantagonist IL-7R antibody to an individual in need of such treatment,thereby preventing or treating one or more symptoms of multiplesclerosis and reducing and/or depleting the naïve and/or activated Tcell populations in the individual. In some embodiments, the reduced ordepleted T cell populations in the individual comprise T_(H)1 and/orT_(H)17 cells. In some embodiments, administration of the antagonistIL-7R antibody does not result in expansion of T_(H)17 cell populationin the individual.

In some embodiments, the antibody can be administered parenterally. Insome embodiments, the individual is a human.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1 depicts the dose-dependent effect of antagonist IL-7R monoclonalantibodies P2D2, P2E11 and HAL403a on IL-7-mediated STAT5phosphorylation in human peripheral blood mononuclear cell (PBMCs). Thex-axis indicates the percentage of CD4+ cells expressing phospho-STAT5(p-STAT).

FIG. 2 depicts the effect of antagonist IL-7R monoclonal antibody 28G9on development of diabetes in non-obese diabetic (NOD) mice.

FIG. 3A depicts the effect of antagonist IL-7R monoclonal antibody 28G9on blood glucose levels (mg/dL) in NOD mice.

FIG. 3B depicts the effect of antagonist IL-7R monoclonal antibody 28G9on body weight (g) in NOD mice.

FIG. 4A depicts the effect of antagonist IL-7R monoclonal antibody 28G9on naïve CD8+ T cell populations in NOD mice. For the x-axis, the totalCD8+ T cell population was set as 100%.

FIG. 4B depicts the effect of antagonist IL-7R monoclonal antibody 28G9on memory CD8+ T cell populations in NOD mice. For the x-axis, the totalCD8+ T cell population was set as 100%.

FIG. 5 depicts the effect of antagonist IL-7R monoclonal antibody 28G9on naive CD4+ T cell population in NOD mice. For the x-axis, the totalCD4+ T cell population was set as 100%.

FIG. 6 depicts the effect of antagonist IL-7R monoclonal antibodies 28B6and 28G9 on clinical severity of EAE animals. Clinical severity of EAEwas assessed daily with a 0 to 5 point scoring system: 0, normal; 1,flaccid tail; 2, partial hind-limb paralysis; 3, total hind-limbparalysis; 4, quadriplegia; 5, moribund state or dead.

FIG. 7 depicts the dose-dependent effect of antagonist IL-7R monoclonalantibody 28G9 on clinical severity of EAE animals.

FIG. 8 depicts the effect of antagonist IL-7R monoclonal antibody 28G9on clinical severity of EAE animals.

FIG. 9 depicts the effect of antagonist IL-7R monoclonal antibody 28G9in animals with established EAE.

FIG. 10 depicts the effect of antagonist IL-7R monoclonal antibody 28G9at lower dose in animals with established EAE.

FIG. 11A depicts the effect of antagonist IL-7R monoclonal antibodies28G9 and 28B6 on CD4 T cell populations from bone marrow (BM), spleen,blood and lymph nodes (LNs) of EAE animals. For the x-axis, the totallymphocyte population was set as 100%.

FIG. 11B depicts the effect of antagonist IL-7R monoclonal antibodies28G9 and 28B6 on CD8 T cell populations from bone marrow (BM), spleen,blood and lymph nodes (LNs) of EAE animals. For the x-axis, the totallymphocyte population was set as 100%.

FIG. 12A depicts the effect of antagonist IL-7R monoclonal antibody 28G9on naive T cell populations from bone marrow, spleen, blood and lymphnodes of EAE animals. For the x-axis, the CD8+ T cell population was setas 100%.

FIG. 12B depicts the effect of antagonist IL-7R monoclonal antibody 28G9on memory T cell populations from bone marrow, spleen, blood and lymphnodes of EAE animals. For the x-axis, the CD8+ T cell population was setas 100%.

FIG. 12C depicts the effect of antagonist IL-7R monoclonal antibody 28G9on activated T cell populations from bone marrow, spleen, blood andlymph nodes of EAE animals. For the x-axis, the CD8+ T cell populationwas set as 100%.

FIG. 13 depicts the effect of antagonist IL-7R monoclonal antibody 28G9on T_(eff) cell (left graph) and T_(reg) cell (right graph) populationsfrom bone marrow, spleen, blood and lymph nodes of EAE animals. For thex-axis, the CD4+ T cell population was set as 100%. “*” indicates P<0.05as compared to control.

FIG. 14 depicts the effect of antagonist IL-7R monoclonal antibody 28G9on blood glucose levels (mg/dL) in high fat diet-induced obesity (DIO)mice.

FIG. 15 depicts the effect of antagonist IL-7R monoclonal antibody 28G9on glucose intolerance in high fat diet-induced obesity (DIO) mice.

DETAILED DESCRIPTION

Disclosed herein are antibodies that antagonize the function of IL-7R,including its interaction with IL-7. Methods of making antagonist IL-7Rantibodies, compositions comprising these antibodies, and methods ofusing these antibodies as a medicament are provided. IL-7R antagonists,e.g., antagonist IL-7R antibodies, can be used to in the preventionand/or treatment of type 2 diabetes, GVHD and autoimmune disorders,including multiple sclerosis (MS), rheumatoid arthritis, type 1diabetes, and lupus.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

DEFINITIONS

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv), single chain(ScFv) and domain antibodies (including, for example, shark and camelidantibodies), and fusion proteins comprising an antibody, and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site. An antibody includes an antibody of any class,such as IgG, IgA, or IgM (or sub-class thereof), and the antibody neednot be of any particular class. Depending on the antibody amino acidsequence of the constant region of its heavy chains, immunoglobulins canbe assigned to different classes. There are five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2. The heavy-chain constant regions that correspond tothe different classes of immunoglobulins are called alpha, delta,epsilon, gamma, and mu, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature 348:552-554, for example.

As used herein, “humanized” antibody refers to forms of non-human (e.g.murine) antibodies that are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Preferably, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat, or rabbit having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, the humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences, but are included to further refineand optimize antibody performance. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region or domain (Fc),typically that of a human immunoglobulin. Preferred are antibodieshaving Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDRH1, CDR H2, or CDR H3) which are altered with respect to the originalantibody, which are also termed one or more CDRs “derived from” one ormore CDRs from the original antibody.

In certain embodiments, definitive delineation of a CDR andidentification of residues comprising the binding site of an antibody isaccomplished by solving the structure of the antibody and/or solving thestructure of the antibody-ligand complex. In certain embodiments, thatcan be accomplished by any of a variety of techniques known to thoseskilled in the art, such as X-ray crystallography. In certainembodiments, various methods of analysis can be employed to identify orapproximate the CDR regions. Examples of such methods include, but arenot limited to, the Kabat definition, the Chothia definition, the AbMdefinition and the contact definition.

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. See, e.g.,Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothiadefinition is similar to the Kabat definition, but the Chothiadefinition takes into account positions of certain structural loopregions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17;Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses anintegrated suite of computer programs produced by Oxford Molecular Groupthat model antibody structure. See, e.g., Martin et al., 1989, Proc NatlAcad Sci (USA), 86:9268-9272; “AbM™, A Computer Program for ModelingVariable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. TheAbM definition models the tertiary structure of an antibody from primarysequence using a combination of knowledge databases and ab initiomethods, such as those described by Samudrala et al., 1999, “Ab InitioProtein Structure Prediction Using a Combined Hierarchical Approach,” inPROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Thecontact definition is based on an analysis of the available complexcrystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol.,5:732-45.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orwhich has been made using any of the techniques for making humanantibodies known to those skilled in the art or disclosed herein. Thisdefinition of a human antibody includes antibodies comprising at leastone human heavy chain polypeptide or at least one human light chainpolypeptide. One such example is an antibody comprising murine lightchain and human heavy chain polypeptides. Human antibodies can beproduced using various techniques known in the art. In one embodiment,the human antibody is selected from a phage library, where that phagelibrary expresses human antibodies (Vaughan et al., 1996, NatureBiotechnology, 14:309-314; Sheets et al., 1998, Proc. Natl. Acad. Sci.(USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381;Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can alsobe made by immunization of animals into which human immunoglobulin locihave been transgenically introduced in place of the endogenous loci,e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016. Alternatively, the human antibody may be prepared byimmortalizing human B lymphocytes that produce an antibody directedagainst a target antigen (such B lymphocytes may be recovered from anindividual or may have been immunized in vitro). See, e.g., Cole et al.Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985;Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No.5,750,373.

As used herein, the term “IL-7R” refers to any form of IL-7R andvariants thereof that retain at least part of the activity of IL-7R.Unless indicated differently, such as by specific reference to humanIL-7R, IL-7R includes all mammalian species of native sequence IL-7R,e.g., human, canine, feline, equine, and bovine.

As used herein, an “IL-7R antagonist” refers to an antibody or moleculethat is able to inhibit IL-7R biological activity and/or downstreampathway(s) mediated by IL-7R signaling, including binding to IL-7,phosphorylation of STAT5, Src kinases, PI3 kinase and Pyk2, andupregulation of Bcl2 protein. Examples of IL-7R antagonists include,without limitation, antagonist IL-7R antibodies, IL-7R siRNA, IL-7RshRNA, and IL-7R antisense oligonucleotides.

Antagonist IL-7R antibodies encompass antibodies that block, antagonize,suppress or reduce (to any degree including significantly) IL-7Rbiological activity, including downstream pathways mediated by IL-7Rsignaling, such interaction with IL-7 and/or elicitation of a cellularresponse to IL-7. For purpose of the present invention, it will beexplicitly understood that the term “antagonist IL-7R antibody”(interchangeably termed “IL-7R antagonist antibody,” “antagonistanti-IL-7R antibody” or “anti-IL-7R antagonist antibody”) encompassesall the previously identified terms, titles, and functional states andcharacteristics whereby the IL-7R itself, an IL-7R biological activity(including but not limited to interaction with IL-7, its ability tomediate any aspect of phosphorylation of STAT5,phosphatidylinositol-3-kinase (PI3K)-Akt pathway activation, p27^(Kip1)downregulation, Bcl-2 upregulation, Rb hyperphosphorylation, and CXCR4upregulation), or the consequences of the biological activity, aresubstantially nullified, decreased, or neutralized in any meaningfuldegree. In some embodiments, an antagonist IL-7R antibody binds IL-7Rand prevents interaction with IL-7. Examples of antagonist IL-7Rantibodies are provided herein.

As used herein a “full antagonist” is an antagonist which, at aneffective concentration, essentially completely blocks a measurableeffect of IL-7R. By a partial antagonist is meant an antagonist that iscapable of partially blocking a measurable effect, but that, even at ahighest concentration is not a full antagonist. By essentiallycompletely is meant at least about 80%, preferably, at least about 90%,more preferably, at least about 95%, and most preferably, at least about98% of the measurable effect is blocked.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength, preferably, relatively short (e.g., 10-100 amino acids). Thechain may be linear or branched, it may comprise modified amino acids,and/or may be interrupted by non-amino acids. The terms also encompassan amino acid chain that has been modified naturally or by intervention;for example, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation or modification,such as conjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. It is understood thatthe polypeptides can occur as single chains or associated chains.

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) also known as hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen-binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-lazikaniet al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

As known in the art a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, an antibody “interacts with” IL-7R when the equilibriumdissociation constant is equal to or less than 20 nM, preferably lessthan about 6 nM, more preferably less than about 1 nM, most preferablyless than about 0.2 nM, as measured by the methods disclosed herein inExample 2.

An epitope that “preferentially binds” or “specifically binds” (usedinterchangeably herein) to an antibody or a polypeptide is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an IL-7R epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other IL-7R epitopes or non-IL-7Repitopes. It is also understood that by reading this definition, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as in Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant regions, CH2 and CH3.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods,4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. “FcR”also includes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,117:587; and Kim et al., 1994, J. Immunol., 24:249).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, binds toan epitope in a manner sufficiently similar to the binding of a secondantibody, or an antigen-binding portion thereof, such that the result ofbinding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. In some embodiments, the variant Fc region has atleast one amino acid substitution compared to a native sequence Fcregion or to the Fc region of a parent polypeptide, e.g. from about oneto about ten amino acid substitutions, and preferably, from about one toabout five amino acid substitutions in a native sequence Fc region or inthe Fc region of the parent polypeptide. The variant Fc region hereinwill preferably possess at least about 80% sequence identity with anative sequence Fc region and/or with an Fc region of a parentpolypeptide, and most preferably, at least about 90% sequence identitytherewith, more preferably, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99% sequenceidentity therewith.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: enhancement of glucose clearance, lowering blood glucoselevels, improving glucose tolerance, reducing incidence of high bloodglucose levels resulting from type 1 or type 2 diabetes, reducingincidence or amelioration of one or more symptoms of rheumatoidarthritis, reducing incidence or amelioration of one or more symptoms ofGVHD, reducing incidence or amelioration of one or more symptoms oflupus, and reducing incidence or amerlioration of one or more symptomsof multiple sclerosis.

“Reducing incidence” means any of reducing severity (which can includereducing need for and/or amount of (e.g., exposure to) other drugsand/or therapies generally used for this condition. As is understood bythose skilled in the art, individuals may vary in terms of theirresponse to treatment, and, as such, for example, a “method of reducingincidence” reflects administering the IL-7R antagonist based on areasonable expectation that such administration may likely cause such areduction in incidence in that particular individual.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an IL-7R antagonist. “Ameliorating”also includes shortening or reduction in duration of a symptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect any one or more beneficial or desired results. For prophylacticuse, beneficial or desired results include eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducingblood glucose levels, reducing incidence or amelioration of one or moresymptoms of type 1 diabetes, type 2 diabetes, rheumatoid arthritis,GVHD, lupus or multiple sclerosis, decreasing the dose of othermedications required to treat the disease, enhancing the effect ofanother medication, and/or delaying the progression of the disease ofpatients. An effective dosage can be administered in one or moreadministrations. For purposes of this invention, an effective dosage ofdrug, compound, or pharmaceutical composition is an amount sufficient toaccomplish prophylactic or therapeutic treatment either directly orindirectly. As is understood in the clinical context, an effectivedosage of a drug, compound, or pharmaceutical composition may or may notbe achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to, farm animals, sportanimals, pets, primates, horses, dogs, cats, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

The term “k_(on)”, as used herein, refers to the rate constant forassociation of an antibody to an antigen. Specifically, the rateconstants (k_(on) and k_(off)) and equilibrium dissociation constantsare measured using Fab antibody fragments (i.e. univalent) and IL-7R.

The term “k_(off)”, as used herein, refers to the rate constant fordissociation of an antibody from the antibody/antigen complex.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Methods for Preventing or Treating Type 2 Diabetes, GVHD and AutoimmuneDisorders

In one aspect, the invention provides a method for treating orpreventing type 2 diabetes in an individual comprising administering tothe individual an effective amount of an IL-7R antagonist such as, forexample, an antagonist IL-7R antibody. In another aspect, the inventionprovides a method for treating or preventing an autoimmune disease, suchas type 1 diabetes, rheumatoid arthritis, lupus or multiple sclerosis,in an individual, the method comprising administering to the individualan effective amount of an IL-7R antagonist. In another aspect, theinvention provides a method for treating or preventing GVHD in anindividual comprising administering to the individual an effectiveamount of an IL-7R antagonist.

In some embodiments, therapeutic administration of the IL-7R antagonistadvantageously results in lower blood glucose level and improved glucosetolerance. In other embodiments, therapeutic administration of the IL-7Rantagonist advantageously maintains blood glucose at desirable levels.

In some embodiments, therapeutic administration of the IL-7R antagonistadvantageously results in reduced incidence and/or amelioration of oneor more symptoms of rheumatoid arthritis including, for example withoutlimitation, joint stiffness, joint swelling, joint pain, and jointredness and warmth.

In some embodiments, therapeutic administration of the IL-7R antagonistadvantageously results in reduced incidence and/or amelioration of oneor more symptoms of lupus including, for example without limitation,fatigue, fever, weight loss, weight gain, joint pain, joint stiffness,joint swelling, malar rash, skin lesions, mouth sores, nose ulcers, hairloss, Raynaud's phenomenon, shortness of breath, chest pain, dry eyes,bruising, anxiety, depression and memory loss.

In some embodiments, therapeutic administration of the IL-7R antagonistadvantageously results in reduced incidence and/or amelioration of oneor more symptoms of multiple sclerosis including, for example withoutlimitation, limb paralysis, tremors, difficulty walking, swallowingdifficulties, blindness, blurring vision, and muscle weakness.

In some embodiments, therapeutic administration of the IL-7R antagonistadvantageously results in reduced incidence and/or amelioration of oneor more symptoms of GVHD including, for example without limitation,abdominal pain, abdominal cramps, fever, jaundice, skin rash, vomiting,weight loss, dry eyes, dry mouth, hair loss, hepatitis, lung disorders,and digestive tract disorders.

In some embodiments, therapeutic administration of the IL-7R antagonistadvantageously results in reduced incidence and/or amelioration of oneor more symptoms of acute GVHD including, for example withoutlimitation, pneumonitis, intestinal inflammation, diarrhea, abdominalpain, abdominal cramps, fever, jaundice, nausea, vomiting, liver damage,skin rash, skin damage, damage to the mucosa, sloughing of the mucosalmembrane, damage to the gastrointestinal tract, weight loss,maculopapular rash, elevated bilirubin levels, morbidity and mortality.

In some embodiments, therapeutic administration of the IL-7R antagonistadvantageously results in reduced incidence and/or amelioration of oneor more symptoms of chronic GVHD including, for example withoutlimitation, dry eyes, dry mouth, hair loss, hepatitis, lung disorders,digestive tract disorders, skin rash, oral ulcer, oral atrophy,onchodystrophy, sicca syndrome, sclerosis, lichen-planus-like lesions,poikiloderma, esophageal webs, fasciitis and bronchiolitis obliterans,and damage to the liver, skin and mucosa, connective tissue, exocrineglands and/or the gastrointestinal tract.

A diabetic individual requiring lower blood glucose levels can betreated with an IL-7R antagonist such as, for example, an antagonistIL-7R antibody. An individual suitable for antibody therapy is selectedusing clinical criteria and prognostic indicators of diabetes that arewell known in the art. An individual at risk of developing diabetes asassessed by known prognostic indicators such as family history, fastingblood glucose levels, or decreased glucose tolerance also warrantsadministration of an IL-7R antagonist. One skilled in the art wouldrecognize or know how to diagnose an individual with diabetes ordisregulated glucose uptake and, depending upon the degree or severityof the disease, can make the appropriate determination of when toadminister the antibody and can also select the most desirable mode ofadministration.

An individual suffering from rheumatoid arthritis can be treated with anIL-7R antagonist such as, for example, an antagonist IL-7R antibody. Anindividual suitable for IL-7R antagonist therapy is selected usingclinical criteria and prognostic indicators of rheumatoid arthritis thatare well known in the art. Diagnosis or assessment of rheumatoidarthritis is well-established in the art. Assessment of severity may beperformed based on measures known in the art, such as the rheumatoidarthritis severity scale (RASS). Bardwell et al., Rheumatology, 2002,41:38-45. In some embodiments, ameliorating, controlling, reducingincidence of, or delaying the development or progression of rheumatoidarthritis and/or symptoms of rheumatoid arthritis is measured by RASS.

An individual suffering from lupus can be treated with an IL-7Rantagonist such as, for example, an antagonist IL-7R antibody. Anindividual suitable for IL-7R antagonist therapy is selected usingclinical criteria and prognostic indicators of lupus that are well knownin the art. One skilled in the art would recognize or know how todiagnose an individual with lupus and, depending upon the degree orseverity of the disease, can make the appropriate determination of whento administer the IL-7R antagonist and can also select the mostdesirable mode of administration.

An individual suffering from multiple sclerosis can be treated with anIL-7R antagonist such as, for example, an antagonist IL-7R antibody. Anindividual suitable for IL-7R antagonist therapy is selected usingclinical criteria and prognostic indicators of multiple sclerosis thatare well known in the art. An individual at risk of developing multiplesclerosis as assessed by known prognostic indicators such as familyhistory or symptom history also warrants administration of an IL-7Rantagonist. One skilled in the art would recognize or know how todiagnose an individual with multiple sclerosis and, depending upon thedegree or severity of the disease, can make the appropriatedetermination of when to administer the IL-7R antagonist and can alsoselect the most desirable mode of administration.

An individual suffering from GVHD can be treated with an IL-7Rantagonist such as, for example, an antagonist IL-7R antibody. Anindividual suitable for IL-7R antagonist therapy is selected usingclinical criteria and prognostic indicators of GVHD that are well knownin the art. Diagnosis or assessment of GVHD is well-established in theart. Tests for GVHD usually depend on the symptoms, but may includegastrointesting endoscopy, with or without a biopsy, liver functionstests (AST, ALP, and bilirubin levels will be increased), livery biopsy,lung x-rays, and/or skin biopsy. Features sufficient to establish thediagnosis of chronic GVHD include, for example without limitation,sclerosis, lichen-planus-like lesions, poikiloderma, esophageal webs,fasciitis and bronchiolitis obliterans (see, e.g., Leet and Flowers,Hematology, January 2008; 2008:134-141). Acute liver GVHD may bemeasured by, for example, the bilirubin level in acute patients. Acuteskin GVHD may result in a diffuse maculopapular rash. Assessment of GVHDseverity may be performed based on measures known in the art. In someembodiments, ameliorating, controlling, reducing incidence of, ordelaying the development or progression of GVHD and/or symptoms of GVHDis measured by overall grade (skin-liver-gut) with each organ stagedindividually from a low of 1 to a high of 4. In some embodiments,ameliorating, controlling, reducing incidence of, or delaying thedevelopment or progression of GVHD and/or symptoms of GVHD is measuredby monitoring body weight.

With respect to all methods described herein, reference to IL-7Rantagonists also includes compositions comprising one or more additionalagents. These compositions may further comprise suitable excipients,such as pharmaceutically acceptable excipients including buffers, whichare well known in the art. The present invention can be used alone or incombination with other conventional methods of treatment.

The IL-7R antagonist can be administered to an individual via anysuitable route. It should be apparent to a person skilled in the artthat the examples described herein are not intended to be limiting butto be illustrative of the techniques available. Accordingly, in someembodiments, the IL-7R antagonist is administered to an individual inaccord with known methods, such as intravenous administration, e.g., asa bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerebrospinal, transdermal,subcutaneous, intra-articular, sublingually, intrasynovial, viainsufflation, intrathecal, oral, inhalation or topical routes.Administration can be systemic, e.g., intravenous administration, orlocalized. Commercially available nebulizers for liquid formulations,including jet nebulizers and ultrasonic nebulizers are useful foradministration. Liquid formulations can be directly nebulized andlyophilized powder can be nebulized after reconstitution. Alternatively,an IL-7R antagonist can be aerosolized using a fluorocarbon formulationand a metered dose inhaler, or inhaled as a lyophilized and milledpowder.

In one embodiment, an IL-7R antagonist is administered via site-specificor targeted local delivery techniques. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources of the IL-7R antagonist or local delivery catheters, such asinfusion catheters, indwelling catheters, or needle catheters, syntheticgrafts, adventitial wraps, shunts and stents or other implantabledevices, site specific carriers, direct injection, or directapplication. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat.No. 5,981,568.

Various formulations of an IL-7R antagonist may be used foradministration. In some embodiments, the IL-7R antagonist may beadministered neat. In some embodiments, IL-7R antagonist and apharmaceutically acceptable excipient may be in various formulations.Pharmaceutically acceptable excipients are known in the art, and arerelatively inert substances that facilitate administration of apharmacologically effective substance. For example, an excipient cangive form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000.

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these agents can be combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history.

An IL-7R antagonist can be administered using any suitable method,including by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). IL-7R antibodies can also beadministered via inhalation, as described herein. Generally, foradministration of IL-7R antibodies, an initial candidate dosage can beabout 2 mg/kg. For the purpose of the present invention, a typical dailydosage might range from about any of 3 μg/kg to 30 μg/kg to 300 μg/kg to3 mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending on the factorsmentioned above. For example, dosage of about 1 mg/kg, about 2.5 mg/kg,about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved, forexample, to reduce blood glucose levels. An exemplary dosing regimencomprises administering an initial dose of about 2 mg/kg, followed by aweekly maintenance dose of about 1 mg/kg of the IL-7R antibody, orfollowed by a maintenance dose of about 1 mg/kg every other week.However, other dosage regimens may be useful, depending on the patternof pharmacokinetic decay that the practitioner wishes to achieve. Forexample, in some embodiments, dosing from one to four times a week iscontemplated. In other embodiments dosing once a month or once everyother month or every three months is contemplated. The progress of thistherapy is easily monitored by conventional techniques and assays. Thedosing regimen (including the IL-7R antagonist(s) used) can vary overtime.

For the purpose of the present invention, the appropriate dosage of anIL-7R antagonist will depend on the IL-7R antagonist (or compositionsthereof) employed, the type and severity of symptoms to be treated,whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, the patient's clearance rate for the administered agent,and the discretion of the attending physician. Typically the clinicianwill administer an IL-7R antagonist until a dosage is reached thatachieves the desired result. Dose and/or frequency can vary over courseof treatment. Empirical considerations, such as the half-life, generallywill contribute to the determination of the dosage. For example,antibodies that are compatible with the human immune system, such ashumanized antibodies or fully human antibodies, may be used to prolonghalf-life of the antibody and to prevent the antibody being attacked bythe host's immune system. Frequency of administration may be determinedand adjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of symptoms, e.g., high blood glucose levels, joint pain,etc. Alternatively, sustained continuous release formulations ofantagonist IL-7R antibodies may be appropriate. Various formulations anddevices for achieving sustained release are known in the art.

In one embodiment, dosages for an IL-7R antagonist may be determinedempirically in individuals who have been given one or moreadministration(s) of an IL-7R antagonist. Individuals are givenincremental dosages of an IL-7R antagonist. To assess efficacy, anindicator of the disease can be followed.

Administration of an IL-7R antagonist in accordance with the method inthe present invention can be continuous or intermittent, depending, forexample, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of an IL-7Rantagonist may be essentially continuous over a preselected period oftime or may be in a series of spaced doses.

In some embodiments, more than one IL-7R antagonist may be present. Atleast one, at least two, at least three, at least four, at least fivedifferent, or more IL-7R antagonists can be present. Generally, thoseIL-7R antagonists may have complementary activities that do notadversely affect each other. For example, one or more of the followingIL-7R antagonists may be used: an antagonist IL-7R antibody, ananti-sense molecule directed to an IL-7R (including an anti-sensemolecule directed to a nucleic acid encoding IL-7R), an IL-7R inhibitorycompound, and an IL-7R structural analog. An IL-7R antagonist can alsobe used in conjunction with other agents that serve to enhance and/orcomplement the effectiveness of the agents.

Therapeutic formulations of the IL-7R antagonist used in accordance withthe present invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington, The Scienceand Practice of Pharmacy 20th Ed. Mack Publishing, 2000), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and may comprise buffers such as phosphate,citrate, and other organic acids; salts such as sodium chloride;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens, such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Liposomes containing the IL-7R antagonist are prepared by methods knownin the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci.USA 82:3688, 1985; Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030,1980; and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes withenhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or ‘poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes. Therapeutic IL-7R antagonist compositions aregenerally placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an IL-7Rantagonist with Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

IL-7R Antagonists

The methods of the invention use an IL-7R antagonist, which refers toany protein, peptide or nucleic acid molecule that blocks, suppresses orreduces (including significantly reduces) IL-7R biological activity,including downstream pathways mediated by IL-7R signaling, such aselicitation of a cellular response to IL-7R. Examples of IL-7Rantagonists include, without limitation, antagonist IL-7R antibodies,IL-7R siRNA, IL-7R shRNA, and IL-7R antisense oligonucleotides.

An IL-7R antagonist should exhibit any one or more of the followingcharacteristics: (a) bind to IL-7R; (b) block IL-7R interaction withIL-7; (c) block or decrease IL-7-mediated STAT5 phosphorylation; (d)decrease blood glucose levels in vivo; (e) increase glucose tolerance invivo; (f) reduce disease severity in experimental autoimmuneencephalomyelitis (EAE); (g) block or decrease PI3K phosphorylation; (h)block or decrease AKT phosphorylation; and (i) block IL-7R interactionwith other yet to be identified factors.

In some embodiments, the IL-7R antagonist is an antagonist IL-7Rantibody. For purposes of this invention, the antagonist IL-7R antibodypreferably reacts with IL-7Rα in a manner that inhibits IL-7R signalingfunction and IL-7 interaction. In some embodiments, the antagonist IL-7Rantibody specifically recognizes primate IL-7R. In some embodiments, theantagonist IL-7R antibody binds primate and rodent IL-7R.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion (e.g., a domain antibody),humanized antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The antibodies may be murine, rat, human, or anyother origin (including chimeric or humanized antibodies).

In some embodiments, the antagonist IL-7R antibody is a monoclonalantibody. The antagonist IL-7R antibody can also be humanized. In otherembodiments, the antibody is human.

In some embodiments, the antibody comprises a modified constant region,such as, for example without limitation, a constant region that hasincreased potential for provoking an immune response. For example, theconstant region may be modified to have increased affinity to an Fcgamma receptor such as, e.g., FcγRI or FcγRIIA.

In some embodiments, the antibody comprises a modified constant region,such as a constant region that is immunologically inert, that is, havinga reduced potential for provoking an immune response. In someembodiments, the constant region is modified as described in Eur. J.Immunol., 1999, 29:2613-2624; PCT Application No. PCT/GB99/01441; and/orUK Patent Application No. 9809951.8. The Fc can be human human IgG1,human IgG2 or human IgG4. The Fc can be human IgG2 containing themutation A330P331 to S330S331 (IgG2Δa), in which the amino acid residuesare numbered with reference to the wild type IgG2 sequence. Eur. J.Immunol., 1999, 29:2613-2624. In some embodiments, the antibodycomprises a constant region of IgG₄ comprising the following mutations(Armour et al., 2003, Molecular Immunology 40 585-593): E233F234L235 toP233V234A235 (IgG4Ac), in which the numbering is with reference to wildtype IgG4. In yet another embodiment, the Fc is human IgG4 E233F234L235to P233V234A235 with deletion G236 (IgG4Ab). In another embodiment theFc is any human IgG4 Fc (IgG4, IgG4Ab or IgG4Ac) containing hingestabilizing mutation S228 to P228 (Aalberse et al., 2002, Immunology105, 9-19). In another embodiment, the Fc can be aglycosylated Fc.

In some embodiments, the constant region is aglycosylated by mutatingthe oligosaccharide attachment residue (such as Asn297) and/or flankingresidues that are part of the glycosylation recognition sequence in theconstant region. In some embodiments, the constant region isaglycosylated for N-linked glycosylation enzymatically. The constantregion may be aglycosylated for N-linked glycosylation enzymatically orby expression in a glycosylation deficient host cell.

The binding affinity (K_(D)) of an antagonist IL-7R antibody to IL-7R(such as human IL-7R) can be about 0.002 to about 200 nM. In someembodiments, the binding affinity is any of about 200 nM, about 100 nM,about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM,or about 2 pM. In some embodiments, the binding affinity is less thanany of about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10nM, about 1 nM, about 500 pM, about 100 pM, about 50 pM, about 20 pM,about 10 pM, about 5 pM, or about 2 pM.

One way of determining binding affinity of antibodies to IL-7R is bymeasuring binding affinity of monofunctional Fab fragments of theantibody. To obtain monofunctional Fab fragments, an antibody (forexample, IgG) can be cleaved with papain or expressed recombinantly. Theaffinity of an IL-7R Fab fragment of an antibody can be determined bysurface plasmon resonance (Biacore™3000™ surface plasmon resonance (SPR)system, Biacore™, INC, Piscataway N.J.) equipped with pre-immobilizedstreptavidin sensor chips (SA) using HBS-EP running buffer (0.01M HEPES,pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Biotinylatedhuman IL-7R (or any other IL-7R) can be diluted into HBS-EP buffer to aconcentration of less than 0.5 μg/mL and injected across the individualchip channels using variable contact times, to achieve two ranges ofantigen density, either 50-200 response units (RU) for detailed kineticstudies or 800-1,000 RU for screening assays. Regeneration studies haveshown that 25 mM NaOH in 25% v/v ethanol effectively removes the boundFab while keeping the activity of IL-7R on the chip for over 200injections. Typically, serial dilutions (spanning concentrations of0.1-10× estimated K_(D)) of purified Fab samples are injected for 1 minat 100 μL/minute and dissociation times of up to 2 hours are allowed.The concentrations of the Fab proteins are determined by ELISA and/orSDS-PAGE electrophoresis using a Fab of known concentration (asdetermined by amino acid analysis) as a standard. Kinetic associationrates (k_(on)) and dissociation rates (k_(off)) are obtainedsimultaneously by fitting the data globally to a 1:1 Langmuir bindingmodel (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). MethodsEnzymology 6. 99-110) using the BIAevaluation program. Equilibriumdissociation constant (K_(D)) values are calculated as k_(off)/k_(on).This protocol is suitable for use in determining binding affinity of anantibody to any IL-7R, including human IL-7R, IL-7R of another mammal(such as mouse IL-7R, rat IL-7R, primate IL-7R), as well as differentforms of IL-7R. Binding affinity of an antibody is generally measured at25° C., but can also be measured at 37° C.

The antagonist IL-7R antibodies may be made by any method known in theart, including the method as provided in Example 1. For the productionof hybridoma cell lines, the route and schedule of immunization of thehost animal are generally in keeping with established and conventionaltechniques for antibody stimulation and production, as further describedherein. General techniques for production of human and mouse antibodiesare known in the art and/or are described herein.

It is contemplated that any mammalian subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human, hybridoma celllines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C., 1975, Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381, 1982. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the IL-7R monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for IL-7R, or a portion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity, if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a human IL-7Rα, or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, the antagonist IL-7R antibody (monoclonal or polyclonal) ofinterest may be sequenced and the polynucleotide sequence may then becloned into a vector for expression or propagation. The sequenceencoding the antibody of interest may be maintained in vector in a hostcell and the host cell can then be expanded and frozen for future use.Production of recombinant monoclonal antibodies in cell culture can becarried out through cloning of antibody genes from B cells by meansknown in the art. See, e.g. Tiller et al., 2008, J. Immunol. Methods329, 112; U.S. Pat. No. 7,314,622.

In an alternative, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. For example, the constant regionmay be engineered to more nearly resemble human constant regions toavoid immune response if the antibody is used in clinical trials andtreatments in humans. It may be desirable to genetically manipulate theantibody sequence to obtain greater affinity to IL-7R and greaterefficacy in inhibiting IL-7R. It will be apparent to one of skill in theart that one or more polynucleotide changes can be made to theantagonist IL-7R antibody and still maintain its binding ability toIL-7R.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains (2) designing thehumanized antibody, i.e., deciding which antibody framework region touse during the humanizing process (3) the actual humanizingmethodologies/techniques and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; and 6,180,370.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent or modified rodent V regionsand their associated CDRs fused to human constant regions. See, forexample, Winter et al. Nature 349:293-299, 1991, Lobuglio et al. Proc.Nat. Acad. Sci. USA 86:4220-4224, 1989, Shaw et al. J Immunol.138:4534-4538, 1987, and Brown et al. Cancer Res. 47:3577-3583, 1987.Other references describe rodent CDRs grafted into a human supportingframework region (FR) prior to fusion with an appropriate human antibodyconstant region. See, for example, Riechmann et al. Nature 332:323-327,1988, Verhoeyen et al. Science 239:1534-1536, 1988, and Jones et al.Nature 321:522-525, 1986. Another reference describes rodent CDRssupported by recombinantly engineered rodent framework regions. See, forexample, European Patent Publication No. 0519596. These “humanized”molecules are designed to minimize unwanted immunological responsetoward rodent anti-human antibody molecules which limits the durationand effectiveness of therapeutic applications of those moieties in humanrecipients. For example, the antibody constant region can be engineeredsuch that it is immunologically inert (e.g., does not trigger complementlysis). See, e.g. PCT Publication No. PCT/GB99/01441; UK PatentApplication No. 9809951.8. Other methods of humanizing antibodies thatmay also be utilized are disclosed by Daugherty et al., Nucl. Acids Res.19:2471-2476, 1991, and in U.S. Pat. Nos. 6,180,377; 6,054,297;5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT PublicationNo. WO 01/27160.

It is apparent that although the above discussion pertains to humanizedantibodies, the general principles discussed are applicable tocustomizing antibodies for use, for example, in dogs, cats, primate,equines and bovines. It is further apparent that one or more aspects ofhumanizing an antibody described herein may be combined, e.g., CDRgrafting, framework mutation and CDR mutation.

In yet another alternative, fully human antibodies may be obtained byusing commercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

In an alternative, antibodies may be made recombinantly and expressedusing any method known in the art. In another alternative, antibodiesmay be made recombinantly by phage display technology. See, for example,U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; andWinter et al., Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, thephage display technology (McCafferty et al., Nature 348:552-553, 1990)can be used to produce human antibodies and antibody fragments in vitro,from immunoglobulin variable (V) domain gene repertoires fromunimmunized donors. According to this technique, antibody V domain genesare cloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, such as M13 or fd, and displayed asfunctional antibody fragments on the surface of the phage particle.Because the filamentous particle contains a single-stranded DNA copy ofthe phage genome, selections based on the functional properties of theantibody also result in selection of the gene encoding the antibodyexhibiting those properties. Thus, the phage mimics some of theproperties of the B cell. Phage display can be performed in a variety offormats; for review see, e.g., Johnson, Kevin S. and Chiswell, David J.,Current Opinion in Structural Biology 3:564-571, 1993. Several sourcesof V-gene segments can be used for phage display. Clackson et al.,Nature 352:624-628, 1991, isolated a diverse array of anti-oxazoloneantibodies from a small random combinatorial library of V genes derivedfrom the spleens of immunized mice. A repertoire of V genes fromunimmunized human donors can be constructed and antibodies to a diversearray of antigens (including self-antigens) can be isolated essentiallyfollowing the techniques described by Mark et al., J. Mol. Biol.222:581-597, 1991, or Griffith et al., EMBO J. 12:725-734, 1993. In anatural immune response, antibody genes accumulate mutations at a highrate (somatic hypermutation). Some of the changes introduced will conferhigher affinity, and B cells displaying high-affinity surfaceimmunoglobulin are preferentially replicated and differentiated duringsubsequent antigen challenge. This natural process can be mimicked byemploying the technique known as “chain shuffling.” (Marks et al.,Bio/Technol. 10:779-783, 1992). In this method, the affinity of“primary” human antibodies obtained by phage display can be improved bysequentially replacing the heavy and light chain V region genes withrepertoires of naturally occurring variants (repertoires) of V domaingenes obtained from unimmunized donors. This technique allows theproduction of antibodies and antibody fragments with affinities in thepM-nM range. A strategy for making very large phage antibody repertoires(also known as “the mother-of-all libraries”) has been described byWaterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene shufflingcan also be used to derive human antibodies from rodent antibodies,where the human antibody has similar affinities and specificities to thestarting rodent antibody. According to this method, which is alsoreferred to as “epitope imprinting”, the heavy or light chain V domaingene of rodent antibodies obtained by phage display technique isreplaced with a repertoire of human V domain genes, creatingrodent-human chimeras. Selection on antigen results in isolation ofhuman variable regions capable of restoring a functional antigen-bindingsite, i.e., the epitope governs (imprints) the choice of partner. Whenthe process is repeated in order to replace the remaining rodent Vdomain, a human antibody is obtained (see PCT Publication No. WO93/06213). Unlike traditional humanization of rodent antibodies by CDRgrafting, this technique provides completely human antibodies, whichhave no framework or CDR residues of rodent origin.

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods231:147, 1999. Methods for making derivatives of antibodies, e.g.,humanized, single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for IL-7R.

The antibodies can be bound to many different carriers. Carriers can beactive and/or inert. Examples of well-known carriers includepolypropylene, polystyrene, polyethylene, dextran, nylon, amylases,glass, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation. In some embodiments, thecarrier comprises a moiety that targets the myocardium.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors (such as expression vectors disclosed in PCTPublication No. WO 87/04462), which are then transfected into host cellssuch as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. See, e.g., PCT Publication No. WO 87/04462. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant regions in place ofthe homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci.81:6851, 1984, or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of an IL-7R monoclonalantibody herein.

Antagonist IL-7R antibodies can be identified or characterized usingmethods known in the art, whereby reduction, amelioration, orneutralization of IL-7R biological activity is detected and/or measured.In some embodiments, an antagonist IL-7R antibody is identified byincubating a candidate agent with IL-7R and monitoring binding and/orattendant reduction or neutralization of a biological activity of IL-7R.The binding assay may be performed with purified IL-7R polypeptide(s),or with cells naturally expressing, or transfected to express, IL-7Rpolypeptide(s). In one embodiment, the binding assay is a competitivebinding assay, where the ability of a candidate antibody to compete witha known IL-7R antagonist for IL-7R binding is evaluated. The assay maybe performed in various formats, including the ELISA format. In otherembodiments, an antagonist IL-7R antibody is identified by incubating acandidate agent with IL-7R and monitoring binding and attendantinhibition of STAT5 phorphorylation.

Following initial identification, the activity of a candidate antagonistIL-7R antibody can be further confirmed and refined by bioassays, knownto test the targeted biological activities. Alternatively, bioassays canbe used to screen candidates directly. Some of the methods foridentifying and characterizing antagonist IL-7R antibodies are describedin detail in the Examples.

Antagonist IL-7R antibodies may be characterized using methods wellknown in the art. For example, one method is to identify the epitope towhich it binds, or “epitope mapping.” There are many methods known inthe art for mapping and characterizing the location of epitopes onproteins, including solving the crystal structure of an antibody-antigencomplex, competition assays, gene fragment expression assays, andsynthetic peptide-based assays, as described, for example, in Chapter 11of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N. Y., 1999. In anadditional example, epitope mapping can be used to determine thesequence to which an antagonist IL-7R antibody binds. Epitope mapping iscommercially available from various sources, for example, PepscanSystems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitopecan be a linear epitope, i.e., contained in a single stretch of aminoacids, or a conformational epitope formed by a three-dimensionalinteraction of amino acids that may not necessarily be contained in asingle stretch. Peptides of varying lengths (e.g., at least 4-6 aminoacids long) can be isolated or synthesized (e.g., recombinantly) andused for binding assays with an antagonist IL-7R antibody. In anotherexample, the epitope to which the antagonist IL-7R antibody binds can bedetermined in a systematic screening by using overlapping peptidesderived from the IL-7R sequence and determining binding by theantagonist IL-7R antibody. According to the gene fragment expressionassays, the open reading frame encoding IL-7R is fragmented eitherrandomly or by specific genetic constructions and the reactivity of theexpressed fragments of IL-7R with the antibody to be tested isdetermined. The gene fragments may, for example, be produced by PCR andthen transcribed and translated into protein in vitro, in the presenceof radioactive amino acids. The binding of the antibody to theradioactively labeled IL-7R fragments is then determined byimmunoprecipitation and gel electrophoresis. Certain epitopes can alsobe identified by using large libraries of random peptide sequencesdisplayed on the surface of phage particles (phage libraries).Alternatively, a defined library of overlapping peptide fragments can betested for binding to the test antibody in simple binding assays. In anadditional example, mutagenesis of an antigen binding domain, domainswapping experiments and alanine scanning mutagenesis can be performedto identify residues required, sufficient, and/or necessary for epitopebinding. For example, domain swapping experiments can be performed usinga mutant IL-7R in which various fragments of the IL-7R polypeptide havebeen replaced (swapped) with sequences from IL-7R from another species,or a closely related, but antigenically distinct protein (such asanother member of the proprotein convertase family). By assessingbinding of the antibody to the mutant IL-7R, the importance of theparticular IL-7R fragment to antibody binding can be assessed.

Yet another method which can be used to characterize an antagonist IL-7Rantibody is to use competition assays with other antibodies known tobind to the same antigen, i.e., various fragments on IL-7R, to determineif the antagonist IL-7R antibody binds to the same epitope as otherantibodies. Competition assays are well known to those of skill in theart.

An expression vector can be used to direct expression of an antagonistIL-7R antibody. One skilled in the art is familiar with administrationof expression vectors to obtain expression of an exogenous protein invivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.Administration of expression vectors includes local or systemicadministration, including injection, oral administration, particle gunor catheterized administration, and topical administration. In anotherembodiment, the expression vector is administered directly to thesympathetic trunk or ganglion, or into a coronary artery, atrium,ventrical, or pericardium.

Targeted delivery of therapeutic compositions containing an expressionvector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer, J. A.Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al.,J. Biol. Chem., 1994, 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA,1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy, 1994, 1:51;Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy,1995, 1:185; and Kaplitt, Nature Genetics, 1994, 6:148). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther., 1992, 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther., 1992,3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 1989,264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol., 1994, 14:2411, andin Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.

In some embodiments, the invention encompasses compositions, includingpharmaceutical compositions, comprising antibodies described herein ormade by the methods and having the characteristics described herein. Asused herein, compositions comprise one or more antibodies thatantagonize the interaction of IL-7R with IL-7, and/or one or morepolynucleotides comprising sequences encoding one or more theseantibodies. These compositions may further comprise suitable excipients,such as pharmaceutically acceptable excipients including buffers, whichare well known in the art.

The antagonist IL-7R antibodies of the invention are characterized byany (one or more) of the following characteristics: (a) bind to IL-7R;(b) block IL-7R interaction with IL-7; (c) block or decreaseIL-7-mediated STAT5 phosphorylation; (d) decrease blood glucose levelsin vivo; (e) improve glucose tolerance in vivo; and (f) reduce diseaseseverity in EAE. Preferably, antagonist IL-7R antibodies have two ormore of these features. More preferably, the antibodies have three ormore of the features. More preferably, the antibodies have four or moreof the features. More preferably, the antibodies have five or more ofthe features. Most preferably, the antibodies have all sixcharacteristics.

Accordingly, the invention provides any of the following, orcompositions (including pharmaceutical compositions) comprising any ofthe following:

(a) an antibody having a partial light chain sequence of (SEQ ID NO: 1)NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDSSHLV FGGGTKLTVLC,(SEQ ID NO: 3) NFMLTQPHSVSESPGKTVTISCTGSSGRIASSYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYASSSLW VFGGGTQLTVLS,(SEQ ID NO: 5) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCMQYDSSHLV FGGGTKLTVLC,(SEQ ID NO: 7) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDFHHLV FGGGTKLTVLC,(SEQ ID NO: 9) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDFHHLV FGGGTKLTVLC,(SEQ ID NO: 11) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCMQYDFHHLV FGGGTKLTVLC,(SEQ ID NO: 44) NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCMQYDFHHLV FGGGTKLTVL, or(SEQ ID NO: 41) NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLV FGGGTKLTVL; and(b) an antibody having a partial heavy chain sequence of (SEQ ID NO: 2)QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSLVGWDGSATYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG DYVFDYWGQGTLVTVSS,(SEQ ID NO: 4) QVTLKESGGGLVQPGGSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTVYLQMNSLRDEDTAVYYCARDI SGGGMDVWGQGTTVTVSS,(SEQ ID NO: 6) QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSLVGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG DYVFNNWGQGTLVTVSS, (SEQ ID NO: 8) QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSLVGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG DYMGDYWGQGTLVTVSS,(SEQ ID NO: 10) QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSLVGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG DYMGNNWGQGTLVTVSS,(SEQ ID NO: 12) QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSLVGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQG DYMGNNWGQGTLVTVSS, or(SEQ ID NO: 40) EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSLVGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQG DYMGNNWGQGTLVTVSS.

TABLE 1 mAb Light Chain Heavy Chain P3A9 NFMLTQPHSVSGSPGKTVTISQVNLRESGGGLVKPGGSLRLSCAAS C TRSSGSIDSSYVQ WYQQRP GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIY EDDQRPS GVPDR LVGWDGSATYYADSVKGRFTISRDNFSGSIDSSSNSASLTISGLVTE TKNLLYLQMNSLRAEDTAVYYCAR Q DEADYYC QSYDSSHLV FGGGGDYVFDY WGQGTLVTVSS TKLTVLC (SEQ ID NO: 2) (SEQ ID NO: 1) P4B3NFMLTQPHSVSESPGKTVTIS QVTLKESGGGLVQPGGSLRLSCAAS C TGSSGRIASSYVQ WYQQRPGFTFS NYGMHWVRQAPGKGLEWVS GSAPTTVIY EDNQRPS GVPDRAISGSGGSTYYADSVKGRFTISRDNS FSGSIDSSSNSASLTISGLKTEKNTVYLQMNSLRDEDTAVYYCAR DIS DEADYYC QSYASSSLWV FGG GGGMDV WGQGTTVTVSSGTQLTVLS (SEQ ID NO: 4) (SEQ ID NO: 3) P2D2 NFMLTQPHSVSGSPGKTVTISQVNLRESGGGLVKPGGSLRLSCAAS C TRSSGSIDSSYVQ WYQQRP GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIY EDDQRPS GVPDR LVGWDGFFTYYADSVKGRFTISRDNFSGSIDSSSNSASLTISGLVTE TKNLLYLQMNSLRAEDTAVYYCAR Q DEADYYC MQYDSSHLV FGGGGDYVFNN WGQGTLVTVSS TKLTVLC (SEQ ID NO: 6) (SEQ ID NO: 5) P2E11NFMLTQPHSVSGSPGKTVTIS QVNLRESGGGLVKPGGSLRLSCAAS C TRSSGSIDSSYVQ WYQQRPGFTFD DSVMHWVRQAPGKGLEWLS GNSPTTVIY EDDQRPS GVPDRLVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLVTETKNLLYLQMNSLRAEDTAVYYCAR Q DEADYYC QSYDFHHLV FGGG GDYMGDY WGQGTLVTVSSTKLTVLC (SEQ ID NO: 8) (SEQ ID NO: 7) HAL NFMLTQPHSVSGSPGKTVTISQVNLRESGGGLVKPGGSLRLSCAAS 403a C TRSSGSIDSSYVQ WYQQRP GFTFDDSVMHWVRQAPGKGLEWLS GNSPTTVIY EDDQRPS GVPDR LVGWDGFFTYYADSVKGRFTISRDNFSGSIDSSSNSASLTISGLVTE TKNLLYLQMNSLRAEDTAVYYCAR Q DEADYYC QSYDFHHLV FGGGGDYMGNN WGQGTLVTVSS TKLTVLC (SEQ ID NO: 10) (SEQ ID NO: 9) HALNFMLTQPHSVSGSPGKTVTIS QVNLRESGGGLVKPGGSLRLSCAAS 403b C TRSSGSIDSSYVQWYQQRP GFTFD DSVMHWVRQAPGKGLEWLS GNSPTTVIY EDDQRPS GVPDRLVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLVTETKNLLYLQMNSLRAEDTAVYYCAR Q DEADYYC MQYDFHHLV FGGG GDYMGNN WGQGTLVTVSSTKLTVLC (SEQ ID NO: 12) (SEQ ID NO: 11) C1GM NFMLTQPHSVSESPGKTVTISEVQLVESGGGLVKPGGSLRLSCAAS C TRSSGSIDSSYVQ WYQQRP GFTFDDSVMHWVRQAPGKGLEWVS GSSPTTVIY EDDQRPS GVPDR LVGWDGFFTYYADSVKGRFTISRDNFSGSIDSSSNSASLTISGLKTE AKNSLYLQMNSLRAEDTAVYYCAR Q DEADYYC QSYDFHHLV FGGGGDYMGNN WGQGTLVTVSS TKLTVL (SEQ ID NO: 40) (SEQ ID NO: 41) C2M3NFMLTQPHSVSESPGKTVTIS EVQLVESGGGLVKPGGSLRLSCAAS C TRSSGSIDSSYVQ WYQQRPGFTFD DSVMHWVRQAPGKGLEWVS GSSPTTVIY EDDQRPS GVPDRLVGWDGFFTYYADSVKGRFTISRDN FSGSIDSSSNSASLTISGLKTEAKNSLYLQMNSLRAEDTAVYYCAR Q DEADYYC MQYDFHHLV FGGG GDYMGNN WGQGTLVTVSSTKLTVL (SEQ ID NO: 40) (SEQ ID NO: 44)In Table 1, the underlined sequences are CDR sequences according toKabat and in bold according to Chothia.

The invention also provides CDR portions of antibodies to IL-7R(including Chothia, Kabat CDRs, and CDR contact regions). Determinationof CDR regions is well within the skill of the art. It is understoodthat in some embodiments, CDRs can be a combination of the Kabat andChothia CDR (also termed “combined CRs” or “extended CDRs”). In someembodiments, the CDRs are the Kabat CDRs. In other embodiments, the CDRsare the Chothia CDRs. In other words, in embodiments with more than oneCDR, the CDRs may be any of Kabat, Chothia, combination CDRs, orcombinations thereof. Table 2 provides examples of CDR sequencesprovided herein.

TABLE 2 Heavy Chain mAb CDRH1 CDRH2 CDRH3 P3A9 DSVMH  LVGWDGSATYYADSVKGQGDYVFDY  (SEQ ID NO: 19) (SEQ ID NO: 21) (SEQ ID NO: 24) P4B3 NYGMH AISGSGGSTYYADSVKG DISGGGMDV (SEQ ID NO: 20) (SEQ ID NO: 22)(SEQ ID NO: 25) P2D2 DSVMH  LVGWDGFFTYYADSVKG QGDYVFNN  (SEQ ID NO: 19)(SEQ ID NO: 23) (SEQ ID NO: 26) P2E11 DSVMH  LVGWDGFFTYYADSVKG QGDYMGDY (SEQ ID NO: 19) (SEQ ID NO: 23) (SEQ ID NO: 27) HAL DSVMH LVGWDGFFTYYADSVKG QGDYMGNN  403a (SEQ ID NO: 19) (SEQ ID NO: 23)(SEQ ID NO: 28) C1GM DSVMH  LVGWDGFFTYYADSVKG QGDYMGNN  (SEQ ID NO: 19) (SEQ ID NO: 23)  (SEQ ID NO: 49); (Kabat);  (Kabat);  GFTFDDS GWDGFF(SEQ ID NO: 46)  (SEQ ID NO: 48) (Chothia); (Chothia); GFTFDDSVMH(SEQ ID NO: 47) (extended) C2M3 DSVMH  LVGWDGFFTYYADSVKG QGDYMGNN (SEQ ID NO: 19) (SEQ ID NO: 23) (SEQ ID NO: 49) HAL DSVMH LVGWDGFFTYYADSVKG QGDYMGNN  403b (SEQ ID NO: 19) (SEQ ID NO: 23)(SEQ ID NO: 28) Heavy X₁X₂VMH,  X₁X₂X₃X₄X₅GX₆X₇TYYADSV X₁X₂X₃X₄X₅X₆X₇X₈,Chain wherein X₁ is  KG, wherein X₁ is  wherein X₁ is  consen- D or N; L or A; X₂ is    Q or D; X₂ is     sus X₂ is S or Y V or I; X₃ is G     G or I; X₃ is D    (SEQ ID NO: 50) or S; X₄ is W or   or S; X₄ is Y or  G; X₅ is D or S;  G; X₅ is M, V or  X₆ is F, G or S;  G; X₆ is G or F;  X₇ is F, A or S X₇ is N, D or M;  (SEQ ID NO: 51) X₈ is N, Y or D(SEQ ID NO: 52) Light Chain mAb CDRL1 CDRL2 CDRL3 P3A9 TRSSGSIDSSYVQEDDQRPS  QSYDSSHLV  (SEQ ID NO: 29) (SEQ ID NO: 31) (SEQ ID NO: 33) P4B3TGSSGRIASSYVQ EDNQRPS  QSYASSSLWV (SEQ ID NO: 30) (SEQ ID NO: 32)(SEQ ID NO: 34) P2D2 TRSSGSIDSSYVQ EDDQRPS  MQYDSSHLV (SEQ ID NO: 29)(SEQ ID NO: 31) (SEQ ID NO: 35) P2E11 TRSSGSIDSSYVQ EDDQRPS  QSYDFHHLV (SEQ ID NO: 29) (SEQ ID NO: 31) (SEQ ID NO: 36) HAL TRSSGSIDSSYVQEDDQRPS  QSYDFHHLV  403a (SEQ ID NO: 29) (SEQ ID NO: 31) (SEQ ID NO: 36)C1GM TRSSGSIDSSYVQ EDDQRPS  QSYDFHHLV  (SEQ ID NO: 29) (SEQ ID NO: 31)(SEQ ID NO: 36) C2M3 TRSSGSIDSSYVQ EDDQRPS  MQYDFHHLV (SEQ ID NO: 29)(SEQ ID NO: 31) (SEQ ID NO: 37) HAL TRSSGSIDSSYVQ EDDQRPS  MQYDFHHLV403b (SEQ ID NO: 29) (SEQ ID NO: 31) (SEQ ID NO: 37) LightTX₁SSGX₂IX₃SSYVQ EDX₁QRPS   X₁X₂YX₃X₄X₅X₆LX₇ Chain wherein  wherein wherein X₁ is  consen- X₁ is R or G;  X₁ is D or N Q or M; X₂ is S  susX₂ is S or R;  (SEQ ID NO: 54) or Q; X₃ is D or  X₃ is D or AA; X₄ is F or  (SEQ ID NO: 53) S; X₅ iS H or   S; X₆ is H orS; X₇ is V or W (SEQ ID NO: 55)

CDR contact regions are regions of an antibody that imbue specificity tothe antibody for an antigen. In general, CDR contact regions include theresidue positions in the CDRs and Vernier zones which are constrained inorder to maintain proper loop structure for the antibody to bind aspecific antigen. See, e.g., Makabe et al., 2007, “ThermodynamicConsequences of Mutations in Vernier Zone Residues of a HumanizedAnti-human Epidermal Growth Factor Receptor Murine Antibody,” Journal ofBiological Chemistry, 283:1156-1166. Determination of CDR contactregions is well within the skill of the art. In some embodiments, anantagonist IL-7R antibody comprises one or more CDR contact regionscomprising an amino acid sequence selected from the group consisting ofFTFDDSVM (SEQ ID NO: 56), GWDGFF (SEQ ID NO: 57), ARX₁X₂X₃X₄ wherein X₁,X₂, X₃, and X₄ can be any amino acid, (SEQ ID NO: 58), SGSIDSSY (SEQ IDNO: 59), EDDQRPSGV (SEQ ID NO: 60), and FHHL (SEQ ID NO: 61).

For any given embodiment containing more than one CDR, the CDRs may beany of Kabat, Chothia, extended, AbM, and/or contact.

The binding affinity (K_(D)) of an antagonist IL-7R antibody to IL-7Rcan be about 0.002 to about 200 nM. In some embodiments, the bindingaffinity is any of about 200 nM, 100 nM, about 50 nM, about 10 nM, about1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM,about 15 pM, about 10 pM, about 5 pM, or about 2 pM. In someembodiments, the binding affinity is less than any of about 250 nM,about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about500 pM, about 100 pM, or about 50 pM.

The invention also provides methods of making any of these antibodies.The antibodies of this invention can be made by procedures known in theart. The polypeptides can be produced by proteolytic or otherdegradation of the antibodies, by recombinant methods (i.e., single orfusion polypeptides) as described above or by chemical synthesis.Polypeptides of the antibodies, especially shorter polypeptides up toabout 50 amino acids, are conveniently made by chemical synthesis.Methods of chemical synthesis are known in the art and are commerciallyavailable. For example, an antibody could be produced by an automatedpolypeptide synthesizer employing the solid phase method. See also, U.S.Pat. Nos. 5,807,715; 4,816,567; and 6,331,415.

In another alternative, the antibodies can be made recombinantly usingprocedures that are well known in the art. In one embodiment, apolynucleotide comprises a sequence encoding the heavy chain and/or thelight chain variable regions of antibody P3A9, P4B3, P2D2, P2E11,HAL403a, HAL403b, C1GM, or C2M3. The sequence encoding the antibody ofinterest may be maintained in a vector in a host cell and the host cellcan then be expanded and frozen for future use. Vectors (includingexpression vectors) and host cells are further described herein.

The invention also encompasses scFv of antibodies of this invention.Single chain variable region fragments are made by linking light and/orheavy chain variable regions by using a short linking peptide (Bird etal., 1988, Science 242:423-426). An example of a linking peptide is(GGGGS)₃ (SEQ ID NO: 13), which bridges approximately 3.5 nm between thecarboxy terminus of one variable region and the amino terminus of theother variable region. Linkers of other sequences have been designed andused (Bird et al., 1988, supra). Linkers should be short, flexiblepolypeptides and preferably comprised of less than about 20 amino acidresidues. Linkers can in turn be modified for additional functions, suchas attachment of drugs or attachment to solid supports. The single chainvariants can be produced either recombinantly or synthetically. Forsynthetic production of scFv, an automated synthesizer can be used. Forrecombinant production of scFv, a suitable plasmid containingpolynucleotide that encodes the scFv can be introduced into a suitablehost cell, either eukaryotic, such as yeast, plant, insect or mammaliancells, or prokaryotic, such as E. coli. Polynucleotides encoding thescFv of interest can be made by routine manipulations such as ligationof polynucleotides. The resultant scFv can be isolated using standardprotein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in whichheavy chain variable (VH) and light chain variable (VL) domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g.,Holliger, P., et al., 1993, Proc. Natl. Acad Sci. USA 90:6444-6448;Poljak, R. J., et al., 1994, Structure 2:1121-1123).

For example, bispecific antibodies, monoclonal antibodies that havebinding specificities for at least two different antigens, can beprepared using the antibodies disclosed herein. Methods for makingbispecific antibodies are known in the art (see, e.g., Suresh et al.,1986, Methods in Enzymology 121:210). Traditionally, the recombinantproduction of bispecific antibodies was based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, with the two heavy chainshaving different specificities (Millstein and Cuello, 1983, Nature 305,537-539).

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantregion sequences. The fusion preferably is with an immunoglobulin heavychain constant region, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690.

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT Publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents and techniques are well known in the art, and are described inU.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods of synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

Humanized antibodies can be made using any methods know in the art. Forexample, four general steps may be used to humanize a monoclonalantibody. These are: (1) determining the nucleotide and predicted aminoacid sequence of the starting antibody light and heavy variable domains(2) designing the humanized antibody, i.e., deciding which antibodyframework region to use during the humanizing process (3) the actualhumanizing methodologies/techniques and (4) the transfection andexpression of the humanized antibody. See, for example, U.S. Pat. Nos.4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;5,693,762; 5,585,089; and 6,180,370.

In the recombinant humanized antibodies, the Fcγ portion can be modifiedto avoid interaction with Fcγ□ receptor and the complement and immunesystems. The techniques for preparation of such antibodies are describedin WO 99/58572. For example, the constant region may be engineered tomore resemble human constant regions to avoid immune response if theantibody is used in clinical trials and treatments in humans. See, forexample, U.S. Pat. Nos. 5,997,867 and 5,866,692.

The invention encompasses modifications to the antibodies andpolypeptides of the invention variants shown in Table 1, includingfunctionally equivalent antibodies which do not significantly affecttheir properties and variants which have enhanced or decreased activityand/or affinity. For example, the amino acid sequence may be mutated toobtain an antibody with the desired binding affinity to IL-7R.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 3 under the heading of“conservative substitutions.” If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 3, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 3 Amino Acid Substitutions Conservative Original ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) Non-polar: Norleucine, Met, Ala, Val, Leu, lie;    -   (2) Polar without charge: Cys, Ser, Thr, Asn, Gin;    -   (3) Acidic (negatively charged): Asp, Glu;    -   (4) Basic (positively charged): Lys, Arg;    -   (5) Residues that influence chain orientation: Gly, Pro; and    -   (6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one ormore amino acids to complete redesign of a region, such as the variableregion. Changes in the variable region can alter binding affinity and/orspecificity. In some embodiments, no more than one to five conservativeamino acid substitutions are made within a CDR domain. In otherembodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR domain. In still other embodiments,the CDR domain is CDR H3 and/or CDR L3.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, MatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example, usingendoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3. In addition, the recombinanthost cell can be genetically engineered to be defective in processingcertain types of polysaccharides. These and similar techniques are wellknown in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

In some embodiments of the invention, the antibody comprises a modifiedconstant region, such as a constant region that has increased affinityto a human Fc gamma receptor, is immunologically inert or partiallyinert, e.g., does not trigger complement mediated lysis, does notstimulate antibody-dependent cell mediated cytotoxicity (ADCC), or doesnot activate microglia; or has reduced activities (compared to theunmodified antibody) in any one or more of the following: triggeringcomplement mediated lysis, stimulating antibody-dependent cell mediatedcytotoxicity (ADCC), or activating microglia. Different modifications ofthe constant region may be used to achieve optimal level and/orcombination of effector functions. See, for example, Morgan et al.,Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000;Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis et al.,Immunological Reviews 163:59-76, 1998. In some embodiments, the constantregion is modified as described in Eur. J. Immunol., 1999, 29:2613-2624;PCT Application No. PCT/GB99/01441; and/or UK Patent Application No.9809951.8. In other embodiments, the antibody comprises a human heavychain IgG2 constant region comprising the following mutations: A330P331to S330S331 (amino acid numbering with reference to the wild type IgG2sequence). Eur. J. Immunol., 1999, 29:2613-2624. In still otherembodiments, the constant region is aglycosylated for N-linkedglycosylation. In some embodiments, the constant region is aglycosylatedfor N-linked glycosylation by mutating the glycosylated amino acidresidue or flanking residues that are part of the N-glycosylationrecognition sequence in the constant region. For example,N-glycosylation site N297 may be mutated to A, Q, K, or H. See, Tao etal., J. Immunology 143: 2595-2601, 1989; and Jefferis et al.,Immunological Reviews 163:59-76, 1998. In some embodiments, the constantregion is aglycosylated for N-linked glycosylation. The constant regionmay be aglycosylated for N-linked glycosylation enzymatically (such asremoving carbohydrate by enzyme PNGase), or by expression in aglycosylation deficient host cell.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572. These antibodiescomprise, in addition to a binding domain directed at the targetmolecule, an effector domain having an amino acid sequence substantiallyhomologous to all or part of a constant region of a human immunoglobulinheavy chain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. In some embodiments, theeffector domain is capable of specifically binding FcRn and/or FcγRIIb.These are typically based on chimeric domains derived from two or morehuman immunoglobulin heavy chain C_(H)2 domains. Antibodies modified inthis manner are particularly suitable for use in chronic antibodytherapy, to avoid inflammatory and other adverse reactions toconventional antibody therapy.

The invention includes affinity matured embodiments. For example,affinity matured antibodies can be produced by procedures known in theart (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al.,1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene,169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson etal., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol.Biol., 226:889-896; and PCT Publication No. WO2004/058184).

The following methods may be used for adjusting the affinity of anantibody and for characterizing a CDR. One way of characterizing a CDRof an antibody and/or altering (such as improving) the binding affinityof a polypeptide, such as an antibody, termed “library scanningmutagenesis”. Generally, library scanning mutagenesis works as follows.One or more amino acid positions in the CDR are replaced with two ormore (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) amino acids using art recognized methods. This generatessmall libraries of clones (in some embodiments, one for every amino acidposition that is analyzed), each with a complexity of two or moremembers (if two or more amino acids are substituted at every position).Generally, the library also includes a clone comprising the native(unsubstituted) amino acid. A small number of clones, e.g., about 20-80clones (depending on the complexity of the library), from each libraryare screened for binding affinity to the target polypeptide (or otherbinding target), and candidates with increased, the same, decreased, orno binding are identified. Methods for determining binding affinity arewell-known in the art. Binding affinity may be determined using Biacore™surface plasmon resonance analysis, which detects differences in bindingaffinity of about 2-fold or greater. Biacore™ is particularly usefulwhen the starting antibody already binds with a relatively highaffinity, for example a K_(D) of about 10 nM or lower. Screening usingBiacore™ surface plasmon resonance is described in the Examples, herein.

Binding affinity may be determined using Kinexa Biocensor, scintillationproximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescencequenching, fluorescence transfer, and/or yeast display. Binding affinitymay also be screened using a suitable bioassay.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position).

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, the library may comprise substitutions in two or morepositions in one CDR. The library may comprise substitution in two ormore positions in two or more CDRs. The library may comprisesubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. The substitution may be prepared usinglow redundancy codons. See, e.g., Table 2 of Balint et al., 1993, Gene137(1):109-18.

The CDR may be CDRH3 and/or CDRL3. The CDR may be one or more of CDRL1,CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR may be a Kabat CDR, aChothia CDR, or an extended CDR.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). Candidates that bind may also be sequenced,thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates(each comprising an amino acid substitution at one or more position ofone or more CDR) with improved binding are also useful for the design ofa second library containing at least the original and substituted aminoacid at each improved CDR position (i.e., amino acid position in the CDRat which a substitution mutant showed improved binding). Preparation,and screening or selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acids (including all 20amino acids), and positions in the CDRs which cannot be changed or whichcan only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library,which includes the improved amino acid, the original amino acid at thatposition, and may further include additional substitutions at thatposition, depending on the complexity of the library that is desired, orpermitted using the desired screening or selection method. In addition,if desired, adjacent amino acid position can be randomized to at leasttwo or more amino acids. Randomization of adjacent amino acids maypermit additional conformational flexibility in the mutant CDR, whichmay in turn, permit or facilitate the introduction of a larger number ofimproving mutations. The library may also comprise substitution atpositions that did not show improved affinity in the first round ofscreening.

The second library is screened or selected for library members withimproved and/or altered binding affinity using any method known in theart, including screening using Biacore™ surface plasmon resonanceanalysis, and selection using any method known in the art for selection,including phage display, yeast display, and ribosome display.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies of this invention. In oneembodiment, a fusion polypeptide is provided that comprises at least 10contiguous amino acids of the variable light chain region shown in SEQID NOs: 1, 3, 5, 7, 9, 11, 41 or 44 and/or at least 10 amino acids ofthe variable heavy chain region shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12or 40. In other embodiments, a fusion polypeptide is provided thatcomprises at least about 10, at least about 15, at least about 20, atleast about 25, or at least about contiguous amino acids of the variablelight chain region and/or at least about 10, at least about 15, at leastabout 20, at least about 25, or at least about 30 contiguous amino acidsof the variable heavy chain region. In another embodiment, the fusionpolypeptide comprises a light chain variable region and/or a heavy chainvariable region, as shown in any of the sequence pairs selected fromamong SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and12, 41 and 40, and 44 and 40. In another embodiment, the fusionpolypeptide comprises one or more CDR(s). In still other embodiments,the fusion polypeptide comprises CDR H3 (VH CDR3) and/or CDR L3 (VLCDR3). For purposes of this invention, a fusion protein contains one ormore antibodies and another amino acid sequence to which it is notattached in the native molecule, for example, a heterologous sequence ora homologous sequence from another region. Exemplary heterologoussequences include, but are not limited to a “tag” such as a FLAG tag ora 6His tag. Tags are well known in the art.

A fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the fusion proteinsof this invention are made by preparing an expressing a polynucleotideencoding them using recombinant methods described herein, although theymay also be prepared by other means known in the art, including, forexample, chemical synthesis.

This invention also provides compositions comprising antibodiesconjugated (for example, linked) to an agent that facilitate coupling toa solid support (such as biotin or avidin). For simplicity, referencewill be made generally to antibodies with the understanding that thesemethods apply to any of the IL-7R binding and/or antagonist embodimentsdescribed herein. Conjugation generally refers to linking thesecomponents as described herein. The linking (which is generally fixingthese components in proximate association at least for administration)can be achieved in any number of ways. For example, a direct reactionbetween an agent and an antibody is possible when each possesses asubstituent capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, on one may becapable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

An antibody or polypeptide of this invention may be linked to a labelingagent such as a fluorescent molecule, a radioactive molecule or anyothers labels known in the art. Labels are known in the art whichgenerally provide (either directly or indirectly) a signal.

The invention also provides compositions (including pharmaceuticalcompositions) and kits comprising, as this disclosure makes clear, anyor all of the antibodies and/or polypeptides described herein.

The invention also provides isolated polynucleotides encoding theantibodies of the invention, and vectors and host cells comprising thepolynucleotide.

Accordingly, the invention provides polynucleotides (or compositions,including pharmaceutical compositions), comprising polynucleotidesencoding any of the following: the antibodies C1GM, C2M3, P3A9, P4B3,P2D2, P2E11, HAL403a and HAL403b, or any fragment or part thereof havingthe ability to antagonize IL-7R.

In another aspect, the invention provides polynucleotides encoding anyof the antibodies (including antibody fragments) and polypeptidesdescribed herein, such as antibodies and polypeptides having impairedeffector function. Polynucleotides can be made and expressed byprocedures known in the art.

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the polynucleotides ofthe invention. In some embodiments, the composition comprises anexpression vector comprising a polynucleotide encoding the antibody asdescribed herein. In other embodiment, the composition comprises anexpression vector comprising a polynucleotide encoding any of theantibodies described herein. In still other embodiments, the compositioncomprises either or both of the polynucleotides shown in SEQ ID NO: 38and SEQ ID NO: 39 below:

C1GM heavy chain variable region (SEQ ID NO: 38)GAGGTCCAGTTAGTGGAGTCTGGGGGAGGCCTGGTCAAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTCTGTCATGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGGTTTCTCTTGTTGGTTGGGATGGTTTTTTTACATACTATGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCGAAGAACTCTCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAAGGGGATTACATGGGGAACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC AC2GM light chain variable region (SEQ ID NO: 39)AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGAATCTCCGGGAAAGACGGTGACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGACAGTTCCTATGTGCAGTGGTACCAGCAGCGCCCGGGCAGCTCCCCCACCACTGTGATCTATGAGGATGACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGAAAACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATTTTCATCATCTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA.

In still other embodiments, the composition comprises either or both ofthe polynucleotides shown in SEQ ID NO: 14 and SEQ ID NO: 15 below:

HAL403a heavy chain variable region (SEQ ID NO: 14)CAGGTCAACTTAAGGGAGTCTGGGGGAGGCCTGGTCAAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTCTGTCATGCACTGGGTCCGTCAAGCTCCGGGGAAGGGTCTGGAGTGGCTCTCTCTTGTTGGTTGGGATGGTTTTTTTACATACTATGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACTTACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGACAAGGGGATTACATGGGGAACAACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTC AHAL403a light chain variable region (SEQ ID NO: 15)AATTTTATGCTGACTCAGCCCCACTCTGTGTCGGGGTCTCCGGGAAAGACGGTGACCATCTCCTGCACCCGCAGCAGTGGCAGCATTGACAGTTCCTATGTGCAGTGGTACCAGCAGCGCCCGGGCAATTCCCCCACCACTGTGATCTATGAGGATGACCAAAGACCCTCTGGGGTCCCTGATCGGTTCTCTGGCTCCATCGACAGCTCCTCCAACTCTGCCTCCCTCACCATCTCTGGACTGGTGACTGAGGACGAGGCTGACTACTACTGTCAGTCTTATGATTTTCATCATCTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTATGT.

Expression vectors, and administration of polynucleotide compositionsare further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a portionthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably, 10 fold higher, even morepreferably, 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to IL-7R or an IL-7Rdomain is effected by an immunoassay or FACS. A cell overexpressing theantibody or protein of interest can be identified.

Compositions

The compositions used in the methods of the invention comprise aneffective amount of an antagonist IL-7R antibody, an antagonist IL-7Rantibody derived polypeptide, or other IL-7R antagonists describedherein. Examples of such compositions, as well as how to formulate, arealso described in an earlier section and below. In some embodiments, thecomposition comprises one or more IL-7R antagonist antibodies. In otherembodiments, the antagonist IL-7R antibody recognizes human IL-7Rα. Inother embodiments, the antagonist IL-7R antibody is a human antibody. Inother embodiments, the antagonist IL-7R antibody is a humanizedantibody. In some embodiments, the antagonist IL-7R antibody comprises aconstant region that is capable of triggering a desired immune response,such as antibody-mediated lysis or ADCC. In other embodiments, theantagonist IL-7R antibody comprises a constant region that does nottrigger an unwanted or undesirable immune response, such asantibody-mediated lysis or ADCC. In other embodiments, the antagonistIL-7R antibody comprises one or more CDR(s) of the antibody (such asone, two, three, four, five, or, in some embodiments, all six CDRs).

It is understood that the compositions can comprise more than oneantagonist IL-7R antibody (e.g., a mixture of antagonist IL-7Rantibodies that recognize different epitopes of IL-7R). Other exemplarycompositions comprise more than one antagonist IL-7R antibody thatrecognize the same epitope(s), or different species of antagonist IL-7Rantibodies that bind to different epitopes of IL-7R.

The composition used in the present invention can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and practice of Pharmacy 20th Ed., 2000,Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations, and may comprise buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutically acceptable excipients arefurther described herein.

The antagonist IL-7R antibody and compositions thereof can also be usedin conjunction with other agents that serve to enhance and/or complementthe effectiveness of the agents.

D. Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising an IL-7Rantagonist (such as, for example, a human antibody) described herein andinstructions for use in accordance with any of the methods of theinvention described herein. Generally, these instructions comprise adescription of administration of the IL-7R antagonist for the abovedescribed therapeutic treatments.

In some embodiments, the IL-7R antagonist is an antagonist IL-7Rantibody. In some embodiments, the antibody is a human antibody. In someembodiments, the antibody is a humanized antibody. In some embodiments,the antibody is a monoclonal antibody. The instructions relating to theuse of an antagonist IL-7R antibody generally include information as todosage, dosing schedule, and route of administration for the intendedtreatment. The containers may be unit doses, bulk packages (e.g.,multi-dose packages) or sub-unit doses. Instructions supplied in thekits of the invention are typically written instructions on a label orpackage insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antagonist IL-7R antibody. The container may furthercomprise a second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

Mutations and Modifications

To express the IL-7R antibodies of the present invention, DNA fragmentsencoding VH and VL regions can first be obtained using any of themethods described above. Various modifications, e.g. mutations,deletions, and/or additions can also be introduced into the DNAsequences using standard methods known to those of skill in the art. Forexample, mutagenesis can be carried out using standard methods, such asPCR-mediated mutagenesis, in which the mutated nucleotides areincorporated into the PCR primers such that the PCR product contains thedesired mutations or site-directed mutagenesis.

One type of substitution, for example, that may be made is to change oneor more cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. Forexample, there can be a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant region of an antibody. In some embodiments,the cysteine is canonical.

The antibodies may also be modified, e.g. in the variable domains of theheavy and/or light chains, e.g., to alter a binding property of theantibody. For example, a mutation may be made in one or more of the CDRregions to increase or decrease the K_(D) of the antibody for IL-7R, toincrease or decrease k_(off), or to alter the binding specificity of theantibody. Techniques in site-directed mutagenesis are well-known in theart. See, e.g., Sambrook et al. and Ausubel et al., supra.

A modification or mutation may also be made in a framework region orconstant region to increase the half-life of an IL-7R antibody. See,e.g., PCT Publication No. WO 00/09560. A mutation in a framework regionor constant region can also be made to alter the immunogenicity of theantibody, to provide a site for covalent or non-covalent binding toanother molecule, or to alter such properties as complement fixation,FcR binding and antibody-dependent cell-mediated cytotoxicity. Accordingto the invention, a single antibody may have mutations in any one ormore of the CDRs or framework regions of the variable domain or in theconstant region.

In a process known as “germlining”, certain amino acids in the VH and VLsequences can be mutated to match those found naturally in germline VHand V_(L) sequences. In particular, the amino acid sequences of theframework regions in the V_(H) and VL sequences can be mutated to matchthe germline sequences to reduce the risk of immunogenicity when theantibody is administered. Germline DNA sequences for human VH and VLgenes are known in the art (see e.g., the “Vbase” human germlinesequence database; see also Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson etal., 1992, J. Mol. Biol. 227:776-798; and Cox et al., 1994, Eur. J.Immunol. 24:827-836.

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant regionof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution is to eliminate asparagine-glycine pairs, which formpotential deamidation sites, by altering one or both of the residues. Inanother example, the C-terminal lysine of the heavy chain of an IL-7Rantibody of the invention can be cleaved. In various embodiments of theinvention, the heavy and light chains of the IL-7R antibodies mayoptionally include a signal sequence.

Once DNA fragments encoding the VH and VL segments of the presentinvention are obtained, these DNA fragments can be further manipulatedby standard recombinant DNA techniques, for example to convert thevariable region genes to full-length antibody chain genes, to Fabfragment genes, or to a scFv gene. In these manipulations, a VL- orVH-encoding DNA fragment is operatively linked to another DNA fragmentencoding another protein, such as an antibody constant region or aflexible linker. The term “operatively linked”, as used in this context,is intended to mean that the two DNA fragments are joined such that theamino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG2 constant region. The IgG constant region sequence can beany of the various alleles or allotypes known to occur among differentindividuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypesrepresent naturally occurring amino acid substitution in the IgG1constant regions. For a Fab fragment heavy chain gene, theV_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region. The CH1 heavy chainconstant region may be derived from any of the heavy chain genes.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region. The kappa constant region maybe any of the various alleles known to occur among differentindividuals, such as Inv(1), Inv(2), and Inv(3). The lambda constantregion may be derived from any of the three lambda genes.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, (SEQ ID NO: 16) such thatthe VH and VL sequences can be expressed as a contiguous single-chainprotein, with the VL and VH regions joined by the flexible linker (Seee.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature348:552-554. The single chain antibody may be monovalent, if only asingle VH and VL are used, bivalent, if two VH and VL are used, orpolyvalent, if more than two VH and VL are used. Bispecific orpolyvalent antibodies may be generated that bind specifically to IL-7Rand to another molecule.

In another embodiment, a fusion antibody or immunoadhesin may be madethat comprises all or a portion of an IL-7R antibody of the inventionlinked to another polypeptide. In another embodiment, only the variabledomains of the IL-7R antibody are linked to the polypeptide. In anotherembodiment, the VH domain of an IL-7R antibody is linked to a firstpolypeptide, while the VL domain of an IL-7R antibody is linked to asecond polypeptide that associates with the first polypeptide in amanner such that the VH and VL domains can interact with one another toform an antigen binding site. In another preferred embodiment, the VHdomain is separated from the VL domain by a linker such that the VH andVL domains can interact with one another. The VH-linker-VL antibody isthen linked to the polypeptide of interest. In addition, fusionantibodies can be created in which two (or more) single-chain antibodiesare linked to one another. This is useful if one wants to create adivalent or polyvalent antibody on a single polypeptide chain, or if onewants to create a bispecific antibody.

In other embodiments, other modified antibodies may be prepared usingIL-7R antibody encoding nucleic acid molecules. For instance, “Kappabodies” (111 et al., 1997, Protein Eng. 10:949-57), “Minibodies” (Martinet al., 1994, EMBO J. 13:5303-9), “Diabodies” (Holliger et al., 1993,Proc. Natl. Acad. Sci. USA 90:6444-6448), or “Janusins” (Traunecker etal., 1991, EMBO J. 10:3655-3659 and Traunecker et al., 1992, Int. J.Cancer (Suppl.) 7:51-52) may be prepared using standard molecularbiological techniques following the teachings of the specification.

Bispecific antibodies or antigen-binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, 1990, Clin. Exp. Immunol.79:315-321, Kostelny et al., 1992, J. Immunol. 148:1547-1553. Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of IL-7R. In some embodiments, the modifiedantibodies described above are prepared using one or more of thevariable domains or CDR regions from a human IL-7R antibody providedherein.

Generation of Antigen-Specific Antibodies

Monoclonal antibodies raised against recombinant mouse IL-7Rα/CD127/Fcchimera (R&D Systems Cat. No. 747-MR), and human antibodies obtained bybiopanning a human naïve antibody library with recombinant IL-7Rα wereevaluated for their ability to bind mouse and human IL-7R. Antibodieswere further screened for their ability to block IL-7-mediated STAT5phosphorylation in human peripheral blood mononuclear cells (PBMCs)and/or monkey PBMCs. This manner of antibody preparation yieldedantagonist antibodies that show blocking of IL-7-mediated STAT5phosphorylation, as shown in Example 1.

Representative materials of the present invention were deposited in theAmerican Type Culture Collection (ATCC) on Feb. 9, 2011. Vector C1GM-VHhaving ATCC Accession No. PTA—is a polynucleotide encoding the C1GMheavy chain variable region, and vector C1GM-VL having ATCC AccessionNo. PTA—is a polynucleotide encoding the C1GM light chain variableregion. The deposits were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and Regulations thereunder (BudapestTreaty). This assures maintenance of a viable curlture of the depositfor 30 years from the date of deposit. The deposit will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Pfizer, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 U.S.C. Section 122 and the Commissioner's rulespursuant thereto (including 37 C.F.R. Section 1.14 with particularreference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

EXAMPLES Example 1 Generating and Screening Antagonist IL-7R Antibodies

This example illustrates the generation and screening of antagonistIL-7R antibodies.

General Procedures for Immunization of Animals for Generating MonoclonalAntibodies:

A 2-month old female Sprague Dawley rat was immunized with 50 ugrecombinant mouse IL-7Rα/CD127/Fc chimera, which includes mouse IL-7Rα(Glu21-Asp239), hCD33 signal peptide (Met 1-Ala 16), and human IgG(Pro100-Lys330) (R&D Systems Cat. No. 747-MR). The antigen was preparedfor immunization by mixing 50 ug antigen in 100 ul PBS with 100 ul SigmaAdjuvant System (Cat. No. S6322). The antigen mixture was vortexed andinjected into the hind footpads and peritoneum on days 0, 2, 5 and 7. Onday 9, 50 ug of antigen without adjuvant was injected intravenously in atotal volume of 150 ul in physiological saline. On day 13, the spleencells were prepared as a single cell suspension and fused withP3x63Ag8.653 mouse myeloma cells following a standard fusion protocolusing 40% PEG 1500 (Boeringer Mannheim Biochemicals #783641). The fusedcells were resuspended in medium containing 18% FBS, 2 mM L-glutamine,pen/strep, hypoxanthine, aminopterin and thymidine (HAT) (Sigma H0262)and 10% hybridoma fusion and cloning supplement (HFCS) (Cat. No. 11 363735 001, Sigma), then plated out in 54 96-well plates at 200 ul/well. Atday 7 after fusion, 150 ul of the medium was aspirated from each well,and the wells were re-fed with 200 ul of fresh medium. At day 11-13,supernatant from each well was tested for antibody to IL-7R and human Fcusing ELISA (described below).

ELISA Screening of Antibodies:

Supernatant media from growing hybridoma clones were screened separatelyfor their ability to bind the recombinant mouse (rm) IL-7R. The assayswere performed with 96-well plates coated overnight with 50 μl of a 1μg/ml solution of the antigen. Fifty-five coated plates were washed 4times with PBS with 0.05% Tween and then 50 ul PBS with 0.5% BSA wasadded to each well. 5 ul from each well of the hybridoma plates wereadded to the assay plates, and the plates were incubated at roomtemperature for 2 hrs to allow binding. Excess reagents were washed fromthe wells between each step with PBS containing 0.05% Tween-20. 50 ulhorseradish peroxidase (HRP) conjugated goat-anti mouse, F(ab′)2, Fcspecific (Jackson #115-036-008) was added to bind to the mouseantibodies bound to the antigen. For detection, 50 ul ABTS,2,2′-Azino-bis(3-ethyl benzothiazoline-6-sulfonic acid) diammonium saltwas added as substrate. The plates were read after 30 mins at 405 nmusing a Molecular Devices THERMOmax™ instrument. Hybridoma clones thatsecreted antibodies that were capable of binding to mouse IL-7R wereselected for further analysis. These positive hybridoma supernatantswere then collected from the hybridoma plates and tested in ELISA assaysagainst human Fc, goat anti rat IgM, and recombinant human (rh) IL-7R.Purified antibodies were then prepared for the antibodies that bound torm IL-7R and antibodies that bound to both rm IL-7R and rh IL-7R.

General Procedures for Generating Fully Human Monoclonal AntibodiesUsing Phage Display:

Anti-human IL-7Rα human antibodies were isolated from a phage displayhuman naïve scFv antibody library (Glanville G. et al., 2009, Proc NatlAcad Sci USA, 106(48):20216-20221) by a series of four rounds ofbio-panning against human IL-7Rα (R&D Systems®). For each round ofpanning, 1 ml IL-7Rα (10 ug/ml in PBS) was coated on an immunotube at 4°C. overnight. The IL-7Rα coated immunotube was washed three times withPBST. 10¹³ phage (1 ml) were added to the immunotube and incubated atroom temperature for 1 hour to allow binding. After binding, theimmunotube was washed eight times with PBST. Bound phage were eluted andused to infect freshly grown TG1 cells. After the fourth round ofpanning, the positive binders were screened against both human IL-7Rαand mouse IL-7R by ELISA. The antibodies binding to both human and mouseIL-7R were further studied for their affinities and blocking function,and antibodies were selected for affinity maturation.

In Vitro Functional Assay:

Hybridoma clones secreting human or mouse IL-7R binding antibodies wereexpanded and supernatants were harvested. Total IgGs were purified fromapproximately 10 ml of the supernatant using protein A beads, dialyzedinto PBS buffer, and the final volume reduced to yield solutions with0.7-1 mg/ml of antibodies. Purified antibodies were then used to testtheir ability to block IL-7-mediated STAT5 phosphorylation in humanPBMCs. For PBMC preparation, whole blood cells were collected throughFicoll gradient. Cells were maintained at 37° C. in 5% CO₂ in conicaltubes (to prevent monocyte/macrophage adherence) for 1-2 h beforestimulation with IL-2.

For the functional screening, human PBMCs were preincubated for 5minutes with test antibodies (10 μg/ml) prior to addition of IL-7. Anon-reactive isotype-matched antibody was used as a negative control(isotype control). Cells were stimulated with human IL-7 (0.1 ng/ml, R&DSystems®) for 15 minutes. To stop the IL-7 stimulation, formaldehyde wasadded directly to the culture medium to a final concentration of 1.6%.Cells were fixed for 15 min at room temperature. Methanol was then addeddirectly to a final concentration of 80%, and samples were stored at 4°C. for 30 minutes to 1 hour before being immunostained. Cells werestained with anti-phospho-STAT5 (p-STAT) antibodies (BD Pharmingen, Y694clone 47) and anti-CD4 antibodies (BD Pharmingen, RPA-T4). Using flowcytometry (LSRII, BD™ Biosciences), CD4+ gated cells were analyzed forp-STAT5 staining. Isotype control was set as 100% of p-STAT.

FIG. 1 illustrates the effect of antagonist IL-7R fully human monoclonalantibodies P2D2 and P2E11, and HAL403a on IL-7-mediated STAT5phosphorylation in human PBMCs. A mouse anti-human IL-7R monoclonalantibody, 13A2F4, was used as a positive control, and a nonreactiveisotype-matched antibody was used as a negative control (isotypecontrol). Human PBMCs were preincubated for 5 minutes with each of thetest antibodies or 13A2F4 at the following concentrations: 0.001, 0.01,0.1, 1, and 10 μg/ml. The isotype control antibody was used at thehighest concentration, 10 μg/ml. Cells were stimulated with human IL-7(0.1 ng/ml) for 15 minutes, then fixed and immunostained as describedabove.

As measured by p-STAT5 staining, human antibodies P2D2, P2E11, HAL403aC1GM, C1GM-2 and C2M3 block human IL-7 mediated signaling in adose-dependent manner (FIG. 1 and data not shown). The isotype controlwas set as 100% p-STAT5 staining. At 10 μg/ml antibody HAL403a blockedSTAT5 phosophorylation very effectively (FIG. 1). C1GM, C1GM-2 and C2M3blocked STAT5 phosophorylation comparable to HAL403a (data not shown).

The amino acid sequence of antagonist IL-7R antibody C1GM heavy chain(SEQ ID NO: 42) is shown below.

(SEQ ID NO: 42) EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSLVGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The amino acid sequence of antagonist IL-7R antibody C1GM light chain(SEQ ID NO: 43) is shown below.

(SEQ ID NO: 43) NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS

The amino acid sequence of antagonist IL-7R antibody C1GM-2 heavy chain(SEQ ID NO: 45) is shown below.

(SEQ ID NO: 45) EVQLVESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWVSLVGWDGFFTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The amino acid sequence of antagonist IL-7R antibody C1GM-2 light chain(SEQ ID NO: 43) is shown below.

(SEQ ID NO: 43) NFMLTQPHSVSESPGKTVTISCTRSSGSIDSSYVQWYQQRPGSSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDFHHLVFGGGTKLTVLQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSC QVTHEGSTVEKTVAPTECS

The amino acid sequence of antagonist IL-7R antibody HAL403a heavy chain(SEQ ID NO: 17) is shown below.

(SEQ ID NO: 17) QVNLRESGGGLVKPGGSLRLSCAASGFTFDDSVMHWVRQAPGKGLEWLSLVGWDGFFTYYADSVKGRFTISRDNTKNLLYLQMNSLRAEDTAVYYCARQGDYMGNNWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The amino acid sequence of antagonist IL-7R antibody HAL403a light chain(SEQ ID NO: 18) is shown below.

(SEQ ID NO: 18) NFMLTQPHSVSGSPGKTVTISCTRSSGSIDSSYVQWYQQRPGNSPTTVIYEDDQRPSGVPDRFSGSIDSSSNSASLTISGLVTEDEADYYCQSYDFHHLVFGGGTKLTVLTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC

Example 2 Determining Antibody Binding Affinity

This example illustrates the determination of antibody binding affinityfor antagonist IL-7R antibodies.

The affinities of antagonist IL-7R antibodies to human IL-7R weremeasured on a surface plasmon resonance Biacore™ 2000 or 3000 biosensorequipped with a research-grade CM5 sensor chip (Biacore™ AB, Uppsala,Sweden—now GE Healthcare). Goat polyclonal anti-human F(ab′)2 fragments(Fc specific) were amine-coupled at saturating levels onto all four flowcells using a standard N-hydroxysuccinimide/ethyldimethylaminopropylcarbodiimide (NHS/EDC) chemistry in HBS-P running buffer (fromBiacore™). The buffer was switched to HBS-P containing 1 mg/mL BSA.Human IL-7R-hFc antigen (R&D systems, Minneapolis, USA) was diluted toabout 30 μg/mL and captured for 3 min at 5 μL/min to give levels ofabout 500-1000 RU per flow cell, leaving one blank to serve as areference channel. Fab, hlgG1, or hlgG2ΔA formats of the antibodies wereinjected in duplicates as a 5-membered 3-fold series starting at 2 μMand a 5-membered 4-fold series starting at 0.4 μM for 3 min at 20-50μL/min. Dissociation was monitored for 5 min. The capture chip wasregenerated after the last injection of each titration with two 30 secpulses of 75 mM phosphoric acid or 10 mM Glycine-HCl pH 1.7. Buffercycles provided blanks for double-referencing the binding response data,which were then fit globally to a simple reversible binding model usingBiaevaluation software v.4.1 to deduce the kinetic association anddissociation rate constants, respectively k_(a) and k_(d). Affinitieswere deduced from their ratio (K_(D)=k_(d)/k_(a)). The results in Table4 show that these antibodies have picomolar or nanomolar affinities forhuman IL-7R.

TABLE 4 K_(on) for IL-7R K_(off) for IL-7R mAb (1/Ms) (1/S) K_(D) forIL-7R (nM) P3A9* 5.60E+04 1.14E−02 204 P4B3* 1.56E+04 3.51E−03 225 P2D2*7.17E+04 8.82E−04 12 P2E11* 1.55E+05 4.97E−04 3 HAL403a* 5.07E+062.86E−04 0.06 HAL403b* 1.39E+06 8.08E−05 0.06 C1GM* 4.85E+06 1.71E−040.04 C1GM** 1.42E+06 4.05E−04 0.286 C1GM-2*** 1.51E+06 4.07E−04 0.270C2M3** 1.41E+06 3.07E−04 0.218 C2M3*** 1.55E+06 3.02E−04 0.195 *Fab;**hIgG1; **hIgG2ΔA

Example 4 Antagonist IL-7R Antibodies Reduce Disease Incidence inNon-Obese Diabetic (NOD) Animals, a Mouse Model for Type 1 Diabetes

This example illustrates the effect of antagonist IL-7R antibodies in amouse model for type 1 diabetes.

To study the in vivo effect of antagonist IL-7R antibodies on thediabetogenic process, a rat anti-mouse antagonist IL-7R antibody, 28G9(Rinat), was tested in NOD mice. NOD mice exhibit a susceptibility tospontaneous development of automimmune insulin dependent diabetesmellitus (IDDM, type 1 diabetes) (Kikutani et al., 1992, Adv. Immunol.51: 285-322). 28G9 blocks IL-7-mediated STAT5 phosphorylation in mousesplenocytes and cross-competes with antagonist IL-7R human antibodiesC1GM, C2M3, HAL403a, HAL403b, P3A9, P4B3, P2D2 and P2E11 in Biacore™

6-8 week old NOD female mice (The Jackson Laboratory) were injectedintraperitoneally (i.p.) weekly starting at 9 weeks old (t=0) witheither 3 or 10 mg/kg body weight of 28G9. PBS or non-reactive isotypematched rat monoclonal antibody (isotype) were used as negativecontrols. The isotype antibody was administered at 10 mg/kg body weight.Mice were monitored two times per week for body weight and bloodglucose. Diabetes was considered established when blood glucose levelwas at or over positive readings, i.e., over 250 mg/dL for twoconsecutive monitorings. The onset of diabetes was dated from the firstof the sequential measurements.

As shown in FIG. 2, none of the mice treated with 28G9 at 10 mg/kgdeveloped diabetes even at 18 weeks of age. In contrast, 75-80% of thePBS and isotype-treated mice developed diabetes (FIG. 2). Although notall mice treated with 28G9 at 3 mg/kg were diabetes-free at the end ofthe study, a significantly reduced diabetes incidence compared to thePBS and isotype controls was observed, demonstrating the inhibitoryeffect of 28G9 on diabetes development was dose-dependent (FIG. 2).Treatment with 28G9 at 10 mg/kg significantly reduced blood glucoselevel compared to isotype or PBS controls (FIG. 3A). Mouse developmentduring antagonist IL-7R antibody treatment was monitored by trackingbody weight and mortality. As shown in FIG. 3B, multiple dosing of 3 or10 mg/kg 28G9 had no significant effect on mouse growth, and nomortality was found at 10 mg/kg. Thus, antagonist IL-7R antibodiesreduce blood glucose levels and inhibit diabetes progression in NODanimals. These results demonstrate that antagonist IL-7R antibodies areeffective in preventing and slowing the progression of type 1 diabetes.

To investigate the effect of antagonist IL-7R antibodies on peripheral Tcell regulation, CD4+ and CD8+ T cells were immunostained for theactivation markers CD44 and CD62L and analyzed by flow cytometry. CD4+and CD8+ T cells were isolated from the peripheral blood of PBS-treated,28G9-treated, or isotype-treated mice. In comparison to the isotypecontrol, the percentage of naive CD8+ T cells(B220−CD8+CD44^(lo)CD62L^(hi)) in mice treated with 28G9 at 10 mg/kg wassignificantly lower, and the percentage of memory CD8+ T cells(B220−CD8+CD44^(hi)CD62L^(hi)) were significantly higher (FIGS. 4A and4B). In contrast, naïve CD4+ T cells (B220−CD4+CD44^(lo)CD62L^(hi)) werenot significantly depleted in antagonist IL-7R antibody treated micecompared to isotype control (FIG. 5). These results indicate thatantagonist IL-7R antibodies reduce blood glucose levels through naïveCD8+ T cell depletion.

Example 5 Antagonist IL-7R Antibodies Delay Onset of Autoimmune Disease

This example illustrates the effect of antagonist IL-7R antibodies in amouse model for multiple sclerosis, experimental autoimmuneencephalomyelitis (EAE).

The STAT5 activation assay was used to identify antagonist IL-7Rantibodies. Spleens from B6 or BALB/c were homogenized in PBS and lysedin ACK lysis buffer (Invitrogen) for 2 min and then filtered through100-μm pore size mesh, pelleted, and resuspended at 5×10⁶ cells/ml inroom temperature to 37° C. RPMI 1640 containing 10% FBS, penicillin (100U/ml), streptomycin (100 μg/ml), and L-glutamine. Cells were maintainedat 37° C. in 5% CO₂ in conical tubes (to prevent monocyte/macrophageadherence) for 1-2 h before stimulation. Cells were preincubated withtest antibody for 5 minutes prior to stimulation with IL-7. Cells weretreated for 15 min with murine IL-7 (mIL-7, 0.1 ng/ml). Formaldehyde wasadded directly to the culture medium to a final concentration of 1.6%,and cells were fixed for 15 min at room temperature. Methanol was thenadded directly to a final concentration of 80%, and samples were storedat 4° C. for 30 min to 1 h before immunostaining. The followingantibodies were used for immunostaining: CD11b-FITC (M1/70),B220-Cy5.5PerCP, TCRβ-FITC, p-STAT5 (Y694, clone 47)-Alexa 647 (BD™Pharmingen). TCRβ+ and CD11b+ cells were stained for phospho-STAT5.IL-7-stimulated STAT5 phosphorylation was observed by gating with TCRβin flow cytometry. A number of antibodies that blocked STAT5phosphorylation were identified, including monoclonal antibodies 28B6and 28G9.

Active EAE was induced in 6- to 8-week-old female B6 mice bysubcutaneous immunization with 100 μg of MOG₃₅₋₅₅ peptide(MEVGWYRSPFSRVVHLYRNGK (SEQ ID NO: 15)) emulsified in CFA containing 1mg/ml of heat-killed Mycobacterium tuberculosis H37RA (Difco) on day 0(see, Steinman and Zamvil, 2006). Additionally, mice received 400 ng ofpertussis toxin (Calbiochem) i.v. in 0.1 ml of PBS on days 0 and 2.Starting on day 7 after MOG immunization, animals were treated twiceweekly with antagonist IL-7R antibody 28B6 (10 mg/kg), antagonist IL-7Rantibody 28G9 (10 mg/kg), or non-reactive isotype-matched antibody (10mg/kg). Compared to isotype control, treatment with either 28G9 or 28B6significantly reduced EAE activity as early as day 15 post immunization(FIG. 6). This result demonstrates that antagonist IL-7R antibodies areeffective in slowing the progression of EAE.

To test whether the antagonist IL-7R antibodies are efficacious in adose-dependent manner, MOG immunized EAE animals were treated witheither 1 or 3 mg/kg of 28G9 at day 7 and day 14 post immunization. Anon-reactive isotype-matched antibody (1 mg/kg) was used as a negativecontrol. In comparison to the isotype control, 28G9 treatment at bothdosage levels reduced EAE severity at disease peak (FIG. 7). Thisinhibitory effect of the antagonist IL-7R antibody lasted for about aweek. This result demonstrates that antagonist IL-7R antibodiesconferred protection at both 1 and 3 mg/kg. In a separate study, MOGimmunized EAE animals were treated weekly with 1, 3 or 10 mg/kg of 28G9starting at day 7. Mice treated with 28G9 at 1 mg/kg showed significantefficacy with no mortality (FIG. 8). These results demonstrate that 1mg/kg of antagonist IL-7R antibody treatment is effective in slowing theprogression of EAE and is well-tolerated.

To investigate antagonist IL-7R antibody efficacy in establisheddisease, MOG immunized EAE animals were treated twice weekly with 28G9at 10 mg/kg starting day 14 after immunization. A non-reactiveisotype-matched antibody (10 mg/kg) was used as a negative control.Compared to the control, treatment with antagonist IL-7R antibodysignificantly reduced EAE severity (FIG. 9). No mortality with theantagonist IL-7R antibody observed. This result demonstrates thatantagonist IL-7R antibodies are effective to ameliorate established,ongoing EAE.

To further determine whether antagonist IL-7R antibodies can reduce EAEat late intervention at lower dose, MOG immunized EAE animals weretreated once weekly with 28G9 at 3 mg/kg starting day 14 afterimmunization. A non-reactive isotype-matched antibody (3 mg/kg) was usedas a negative control. Compared to the control, a highly significantreduction of disease severity was observed with antagonist IL-7Rantibody treatment (FIG. 10). This result demonstrates that antagonistIL-7R antibody treatment is effective to reduce disease activity even atlate intervention and lower dose.

Example 6 Immunological Changes after Antagonist IL-7R Antibody Therapyin Autoimmune Disease

This example illustrates immunological changes in EAE mice afterantagonist IL-7R antibody treatment.

To gain insight into the mechanisms by which antagonist IL-7R antibodyacts to ameliorate EAE in the mouse model, lymphocyte populations fromtreated and control animals were analyzed by immunostaining and flowcytometry. For the immunological studies in this example, MOG immunizedEAE animals were treated weekly with antagonist IL-7R antibody 28G9 (10mg/kg), 28B6 (10 mg/kg) or vehicle (non-reactive isotype-matchedantibody, 10 mg/kg). In selected studies, a group of MOG immunized EAEanimals were treated weekly with 28B6 (10 mg/kg). Animals weresacrificed on day 21 after immunization, and central lymphoid organswere collected. Lymphocytes were prepared from the organs and stained asdescribed below. Immunostained lymphocytes were analyzed by flowcytometry.

T cell populations in the BM, spleen, blood and thymus from EAE animalstreated with antagonist IL-7R antibodies were significantly reducedcompared to vehicle controls. As shown in FIG. 11, both CD4 T cell (FIG.11A) and CD8 T cell (FIG. 11B) populations from BM, spleen, blood andlymph nodes were significantly reduced in antagonist IL-7R antibodytreated EAE animals. This is consistent with the role of IL-7R in bothCD4 and CD8 T cell development. However, B cell populations were notsignificantly reduced in all of peripheral lymphoid organs. This resultdiffers from the mouse genetic data from the IL-7R knockout, which lacksboth T and B cells.

Because IL-7R signaling is critical for naïve T cell survival and formemory T cell proliferation, the effect of antagonist IL-7R antibodiesin the regulation of peripheral T cells was analyzed by immunostainingusing activation markers CD44 and CD62L. CD44^(lo)CD62L^(hi) representsnaïve T cells, CD44^(hi)CD62L^(lo) represents activated T cells andCD44^(hi)CD62L^(hi) represent memory T cells. Compared to vehicle(nonreactive isotype-matched antibody) treated animals, antagonist IL-7Rantibody treated mice had significantly depleted naïve T cell andactivated T cell populations (FIGS. 12A and 12C). However, memory T cellpopulations were not significantly depleted (FIG. 12B). This selectivedepletion of naïve and activated T cell populations may provide benefitin that naïve T cell depletion can block nascent autoAg-specific T cellactivation, in turn preventing EAE. Memory T cells are not depleted, andthus, anti-infection immunity is preserved.

A reduction of NK cells in antagonist IL-7R antibody treated EAE animalswas not observed. A slight increase in the percent of NK cells wasobserved, presumably due to the decreased percent of CD4 and CD8 Tcells. This data is consistent with the observation that IL-7/IL-7Rsignaling regulates T cell, but not for NK cell, development.

To determine the effect of antagonist IL-7R antibody treatment onT_(reg) cell population in EAE animals, lymphocytes were stained forFoxp3 to identify T_(reg) cells and MOG-MHC class II (I-Ab) tetramer todetect MOG-specific T cells. The population of MOG-specific CD4+ T_(eff)cells detected in lymph nodes from 28G9 treated EAE animals was similarto that of control (nonreactive isotype-matched antibody-treated)animals (FIG. 13, left graph). However, an increase in T_(reg) cellpopulation was observed with 28G9 treatment (FIG. 13, right graph).These results demonstrate that treatment of EAE animals with antagonistIL-7R antibody results in an increase of T_(reg) cell population.Advantageously, antagonist IL-7R antibody treatment may not developother inflammatory disease, a side effect observed with IL-2Rα antibodytherapy.

Lymphocyte Preparation and Immunofluorescent Staining

For the above studies, single-cell leukocyte suspensions from spleens,peripheral lymph nodes (paired axillary, bronchial and inguinal), thymusand bilateral femurs bone marrow (BM) were generated by gentledissection. Mononuclear cells from the central nervous system (CNS) wereisolated after cardiac perfusion with PBS. Briefly, CNS tissues weredigested with collagenase D (2.5 mg/ml; Roche Diagnostics) and DNaseI (1mg/ml; Roche Diagnostics) at 37° C. for 45 minutes. Mononuclear cellswere isolated by passing the tissue through 70-μm cell strainers (BDBiosciences), followed by Percoll gradient (70%/37%) centrifugation.Lymphocytes were collected from the 37%/70% interface and washed. Thefollowing antibodies were used for immunostaining: FITC-, PE- orPE-Cy5-conjugated CD3 (17A2), CD4 (H129.19), CD8 (53-6.7), CD62L(MEL14), CD44 (IM7), B220 (H1.2F3), IgM (11/41), DX5 (CD49b) (all fromBD Biosciences). For intracellular cytokine staining, lymphocytes werestimulated in vitro with phorbol 12-myristate 13-acetate (20 ng/ml;Sigma-Aldrich) and ionomycin (1 μg/ml; Sigma-Aldrich) in the presence ofGolgiStop™ (monensin) (5 ug/ml) for 5 hours before staining.MOG₃₈₋₄₉/IAb tetramer and control tetramer (CLIP/IAb) were constructedand supplied by the NIH Tetramer Core Facility. Background staining wasassessed using nonreactive, isotype-matched control mAbs. For 2- or3-color immunofluorescence analysis, single-cell suspensions (10⁶ cells)were stained at 4° C. using predetermined optimal concentrations of mAbfor 20 minutes. For tetramer staining, lymphocytes were stained for 3hours at 37° C.

Example 7 Antagonist IL-7R Antibodies Ameliorate Glucose Intolerance inDiet-Induced Obesity (DIO) Animals

This example illustrates the effect of antagonist IL-7R antibodies in amouse model for type 2 diabetes.

To study the in vivo effect of antagonist IL-7R antibodies onpre-established adipose inflammation in DIO mice, C57BL/6 male mice (TheJackson Laboratory) were fed a high fat diet (HFD, D12492, 60 Kcal %fat, Research Diets) beginning at six weeks old. After ten weeks of highfat diet, the 16-week-old obese mice were randomly assigned to groupsfor i.p. administration of antagonist IL-7R antibody 28G9 (10 mg/kg),PBS, or nonreactive isotype-matched control antibody (10 mg/kg). Fourdays after antibody treatment, the mice were subject to a glucosetolerance test (i.p. 1 g/kg, after 16 hr fasting) to assess glucoseintolerance. Table 5 shows the average body weight and glucose levelsfor each of the treated groups (PBS-, isotype- or 28G9-treated mice).The animals in each group had similar body weight.

TABLE 5 Glucose (mg/dL) Treatment Body weight (g) non-fasting/fastingPBS 44.4 232/152.6 isotype 42.3 233/183.6 28G9 41.4 229/122.2

The results of the glucose tolerance test are depicted in FIG. 14.Glucose intolerance induced by high fat diet was ameliorated byantagonist IL-7R antibody treatment. In the glucose tolerance test, DIOmice treated with 28G9 had significantly lower blood glucose levelscompared to mice treated with isotype or PBS (FIG. 14). This resultdemonstrates that antagonist IL-7R antibodies are efficacious in ananimal model for type 2 diabetes.

Example 8 Antagonist IL-7R Antibodies Reduce Disease Severity in a MouseModel for Rheumatoid Arthritis

This example illustrates the effect of antagonist IL-7R antibodies in amouse model for rheumatoid arthritis (RA).

Collagen induced arthritis (CIA) is a widely used animal model sharingmany pathological and histological similarities with RA. To study the invivo effect of antagonist IL-7R antibodies on CIA, 6-8 week old maleB10.RIII mice (stock #000457, The Jackson Laboratory) were immunizedwith 150 ug of Type II collagen (Elastin Products) emulsified inFreund's complete adjuvant containing 4 mg/ml heat-killed Mycobacteriumtuberculosis H37RA (Difco) on day 0 and day 15. Mice were injected i.p.with 1, 3 or 10 mg/kg of antagonist IL-7R antibody 28G9 or nonreactiveisotype-matched control antibodies on day −1, day 1, day 8, day 15 andday 22.

Clinical signs of CIA were assessed daily with a 0 to 5 point scoringsystem: 0, normal; 1, hind or fore paw joint affected or minimal diffuseerythema and swelling; 2, hind or fore paw joints affected or milddiffuse erythema and swelling; 3, hind or fore paw joints affected ormoderate diffuse erythema and swelling; 4, Marked diffuse erythema andswelling; 5, diffuse erythema and severe swelling entire paw, unable toflex digits. Treatment with 28G9 at 3 mg/kg significantly reduced theseverity of CIA in CII-immunized mice as compared to isotype control(FIG. 15). Treatment with 28G9 at 1 mg/kg did not show significantreduction. This result demonstrates that antagonist IL-7R antibodies areeffective in slowing disease progression in an animal model forrheumatoid arthritis.

Example 9 Antagonist IL-7R Antibodies Reduce Disease Severity in a MouseModel for Established EAE

This example illustrates efficicacy of antagonist IL-7R antibodies in amouse model for established EAE.

EAE was induced in SJL/J mice by immunization with 200 μg ofPLP(p139-151) dissolved in complete Freund's adjuvant containing 4 mg/mlof heat-killed Mycobacterium tuberculosis H37Rα (Difco Laboratories).Mice were examined daily for bodyweight measurements and clinical signsof EAE and scored as follows: 0, no paralysis; 1, loss of tail tone; 2,hindlimb weakness; 3, hindlimb paralysis; 4, hindlimb and forelimbparalysis; 5, moribund or dead.

Mice having a EAE clinical score of 2-3 were treated with 28G9 (10mg/kg, i.p.), SB/14 (10 mg/kg, i.p.) or control IgG (10 mg/kg, i.p.)once a week for 2 weeks (on days 0, 7 and 14). 28G9 is rat IgG1 antibodyand SB/14 (BD Biosciences) is a rat IgG2a antibody. Clinical scores weremonitored daily until day 61.

By day 7, mice treated with 28G9 had clinical scores lower than 2 (N=7).The mice treated with 28G9 maintained clinical scores of about 2 untilthe end of the study (day 61). In comparison, the control IgG-treatedanimals had clinical scores between 3 and 4 throughout the study. Noreduction of disease severity was observed with SB/14 treatment comparedto the control.

A highly significant reduction of disease severity was observed in 28G9antagonist IL-7R antibody treated animals. These results demonstratethat antagonist IL-7R antibodies are effective in reducing diseaseseverity in established autoimmune disease.

Example 10 Antagonist IL-7R Antibodies Reduce Blood Glucose Levels inAnimals with Newly Onset Diabetes

This example illustrates the efficacy of antagonist IL-7R antibodies inreversing newly onset diabetes in a mouse model for type 1 diabetes.

A panel of antagonist IL-7R antibodies were tested in a mouse model fortype 1 diabetes. 28G9 is a rat IgG1 monoclonal antibody, 28G9-mIgG2a isan antibody having the 28G9 variable regions with mouse IgG2a constantregion, and agly-28G9 is an aglycosylated antibody having the 28G9variable regions with mouse IgG2a N297A. For construction and expressionof 28G9-mIgG2a, the VH and Vk gene of rat monoclonal antibody 28G9 wereamplified by PCR, cloned into pARC mouse IgG2a and pARC mouse kappamammalian expression vectors, and cotransfected into 293F cells byLipofectamin™ (Invitrogen™). After 5 days of post-transfection, theculture media was harvested and the 28G9 mouse IgG2a was purified byusing Mabselect™ (GE) resin. For construction and expression ofagly-28G9, the VH of rat 28G9 was cloned into an engineered pARC mouseIgG2a vector in which Asn-297 of the CH2 domain was replaced by Ala(pARC mouse IgG2a-N297A). An aglycosylated m28G9 (agly-28G9) wasobtained by cotransfection of 293F cells with pARC mouse IgG2a-N297A andpARC-28G9 mouse kappa vector.

Spontaneous new onset diabetic NOD mice (i.e., two consecutive bloodglucose concentrations over 250 mg/dl) were treated i.p. weekly with28G9-mIgG2a (10 mg/kg, i.p.), 28G9 (10 mg/kg, i.p.), agly-28G9 (10mg/kg, i.p.) or control IgG (10 mg/kg, i.p.). Blood glucose levels weremonitored daily for 140 days after disease onset. In mice treated with28G9-mIgG2a, 100% remission was observed. In the 28G9-mIgG2a treated NODmice, blood glucose levels were maintained below 250 mg/dl with weekly28G9-mIgG2a injections. 28G9 also showed some efficicacy in reducingblood glucose levels compared to control IgG. Agly-28G9-treated andcontrol IgG-treated mice had blood glucose levels of greater than 250mg/dl throughout the study. These results demonstrate that 28G9-mIgG2aantagonist IL-7R antibody is highly effective in reducing blood glucoselevels in mice with established type 1 diabetes.

In a separate study, spontaneous new onset diabetic NOD mice weretreated weekly, beginning at disease onset, with 28G9-mIgG2a (10 mg/kgi.p.) for the number of doses indicated in Table 6. Blood glucose levelswere monitored daily.

TABLE 6 Age at disease Total # of Mouse onset (days) doses Daysdiabetes-free 1 140 3 117 2 190 2 89 3 210 3 145The results shown in Table 6 indicate that two doses of antagonist IL-7Rantibody were sufficient to maintain blood glucose levels lower than 250mg/dL for up to 89 days. Three doses of antagonist IL-7R antibody weresufficient to maintain blood glucose levels lower than 250 mg/dL for atleast 117 days. In this study, blood glucose levels maintained at lowerthan 250 mg/dL were observed for up to five months post-antagonist IL-7Rantibody treatment.

The results of the studies described above demonstrate that antagonistIL-7R antibody 28G9-mIgG2a was highly effective in reducing bloodglucose levels in animals with newly onset diabetes. Furthermore, micetreated with just two or three doses of antagonist IL-7R antibodiesmaintained blood glucose levels lower than 250 mg/dL for several monthsafter antibody was administered.

Example 11 Antagonist IL-7R Antibodies Reduce Disease Severity in MouseModels for Graft-Versus-Host Disease (GVHD)

This example illustrates the effect of antagonist IL-7R antibodies inmouse models for acute and chronic graft-versus-host disease (GVHD).

Acute GVHD

For the mouse model of acute GVHD, 10×10⁶ human PBMC (freshly isolated)were injected into non-irradiated NOD.SCID IL2Rγ−/− mice (The JacksonLaboratory, 8-12 weeks old). 14 days after injection, the mice aretreated with 10 mg/kg antagonist antagonist IL-7R fully human IgG1antibody HAL403b (n=10) or isotype control (n=10) once weekly. Clinicalsigns of GVHD and body weight are monitored twice a week. Forty dayspost-treatment, 100% of antagonist IL-7R antibody-treated animalsremained alive, in contrast to only 50% of isotype control-treatedanimals survived. This result indicates that antagonist IL-7R antibodiesare effective in reducing mortality rate in an animal model for acuteGVHD.

Chronic GVHD

For the chronic GVHD mouse model, human cord blood cells containing asmall (1-5%) percentage of CD3+ T cells are transplanted into newbornirradiated NOD.SCID IL2Rγ−/− mice. Briefly, human CD34+ cord blood(AllCells, LLC, Emeryville, Calif.) was depleted of CD3+ T cells usinghuman CD3 selection beads (Miltenyi Biotec GmBH, Germany, CAT#130-050-101) For the transplantation, about 300,000 to 400,000 CD34+cells containing about 1-5% CD3+ T cells (in a volume of 50 μl) areintracardially injected per newborn irradiated NOD.SCID IL2Rγ−/− mouse(The Jackson Laboratory). cGVHD develops 16-20 weekspost-transplantation.

Beginning at 24 weeks of age, mice with cGVHD are injected with 10 mg/kgantagonist IL-7R fully human IgG1 antibody HAL403b (n=4) or PBS (n=4)once weekly until sacrifice.

Mice are sacrificed at about 28-32 weeks old, after about 4 to 8 weeksof antagonist IL-7R antibody or PBS treatment. Mice treated withantagonist IL-7R fully human IgG1 antibody had significantly less hairloss than mice injected with PBS. Histologic analysis showed kidneys ofPBS-treated mice were generally more severely affected than kidneys ofantagonist IL-7R antibody-treated mice. For example, kidneys of control(PBS-treated) mice had markedly thickened capillary loops with increasedamounts of eosinophilic material. In contrast, kidneys of mice treatedwith antagonist IL-7R antibody had mildly thickened capillary loops withincreased amount of eosinophilic material. In addition, kidneys of micetreated with antagonist IL-7R antibody had fewer dilated tubulescompared to kidneys of mice treated with isotype control, which showedmany dilated tubules. Lung histology revealed substantially reducedbronchial associated lymphoid tissue (BALT) in lungs of mice treatedwith antagonist IL-7R antibody compared to lungs of control mice, whichhad some BALT present. Severe lymphoid atrophy was observed in spleen ofmice treated with antagonist IL-7 R antibody, compared to the mild tomoderate change in spleen of mice treated with PBS.

These results indicate that antagonist IL-7R antibodies are effective inreducing disease severity in an animal model for chronic GVHD.

Example 12 Antagonist IL-7R Antibodies Reduce Disease Severity in aMouse Model for Lupus

This example illustrates the effect of antagonist IL-7R antibodies in amouse model for lupus.

For the mouse model of lupus, MRL/MpJ-Fas^(lpr)/J mice (The JacksonLaboratory) were used. Commonly referred to as lpr mutants, these miceare homozygous for the lymphoproliferation spontaneous mutation(Fas^(lpr)) and show systemic autoimmunity, massive lymphadenopathyassociated with proliferation of aberrant T cells, arthritis, and immunecomplex glomerulonephrosis. As such, the MRL/MpJ-Fas^(lpr)/J mice areuseful as a model for systemic lupus erythematosus.

Beginning at the time of disease onset, mice were dosed i.p. weekly with1, 3, or 10 mg/kg 28G9-mIgG2a antagonist IL-7R antibody (see Example10), 1 mg/kg agly-28G9 antagonist IL-7R antibody, an isotype control IgG(negative control) or cyclophosphamide (positive control). For eachtreatment group, ten mice were used (n=10). Disease severity wasmonitored by measuring proteinuria levels, activity levels, andassessing the righting reflex. In assessing the righting reflex, micethat failed to right themselves within 30 seconds were sacrificed.Survival rate is summarized in Table 7 below.

TABLE 7 Survival rate at Survival rate at 8 weeks after 12 weeks afterTreatment disease onset disease onset 1 mg/kg 28G9-mIgG2a 60% 50% 3mg/kg 28G9-mIgG2a 80% 70% 10 mg/kg 28G9-mIgG2a 90% 80% 1 mg/kg agly-28G9100%  100%  isotype control IgG 60% 60% (negative control)cyclophosphamide 80% 80% (positive control)

As shown in Table 7, mice treated with 1 mg/kg agly-28G9, 3 mg/kg28G9-mIgG2a or 10 mg/kg 28G9-mIgG2a had an increased survival ratecompared to mice treated with isotype control IgG. These resultsindicate that antagonist IL-7R antibodies are effective in reducingdisease severity in an animal model for lupus.

Example 13 Epitope Mapping/Binding of Antagonist IL-7R Antibodies

This example illustrates structure-guided mutagenesis to map antibodybinding epitopes.

Based on the crystal structure of the IL-7/IL-7Rα complex and the likelyinvolvement of certain residues in IL-7 binding (McElroy et al., 2009,Structure, 17(1):54-65, twenty-three IL-7Rα surface-residue mutants(N29D, V58S, E59R, R66N, K77S, L80S, 182S, K84S, K100S, T105A, N131S,Q136K, K138S, Y139F, K141S, M144A, D190S, H191N, Y192A, Y192F, K194S,K194A and F196S) were chosen for mutation to map the antibody bindingepitopes. The numbering for the mutants (i.e., N29D, V58S, E59R, etc.)follows the convention of post-processed protein wherein the first 20amino acids are not counted.

A panel of IL-7Rα single point mutants (his-tagged) were prepared asfollows. The twenty-three IL-7Rα single point mutants described abovewere generated from the previously described wild-type DNA construct(McElroy et al., 2009, supra) using standard DNA techniques. The mutantproteins were expressed using transient transfection in HEK293T cellsand secreted into the cell media. The mutant proteins were purified byNi²⁺ column chromatography. Protein concentrations were measured byspectrophotometry (NanoDrop™).

Interaction analysis of IL-7Rα was performed at 25° C. using asurface-plasmon resonance-based ProteOn™ XPR36 biosensor equipped with aGLM sensor chip (Bio-Rad, Hercules, Calif., USA). HBST running buffer(10 mM Hepes pH7.4, 150 mM NaCl, 0.05% v/v Tween-20) was usedthroughout. Full-length IL-7R antibodies (HAL403a or HAL403b) wereamine-coupled onto separate “vertical” channels of the chip via standardEDC/sulfo-NHS-mediated chemistry to levels of about 2000-5000 RU. Thepanel of IL-7Rα mutants (including wild-type IL-7Rα) was screened in the“horizontal” direction at 100 nM using association and dissociationphases of 3 and 10 mins respectively at 30 uL/min. Surfaces wereregenerated with 2/1 v/v Pierce immunopure elution buffer (pH2.8)/4MNaCl. Most injections were duplicated to confirm that the assay wasreproducible.

Table 8 summarizes the impact of the single point mutations in theIL-7Rα mutants on antibody binding compared to wild-type IL-7Rα.

TABLE 8 Impact on antibody (HAL403a IL-7Rα mutant or HAL403b) bindingI82S Highly impaired K84S, K100S, T105A, Impaired Y192A D190S, H191N,K194S Slightly impaired N29D, V58S, E59R, R66N, None K77S, L80S, N131S,Q136K, K138S, Y139F, K141S, M144A, Y192F, K194A, F196S, (wild-type)

The IL-7Rα mutants displaying weakened antibody binding compared towild-type IL-7Rα were identified as having a point mutation at a residueinvolved in mAb binding. The binding residues of IL-7Rα to antibodyHAL403a in descending order of mutant effects were identified asfollows: 182 (high impact on binding), K84 (medium impact), K100 (mediumimpact), T105 (medium impact), Y192 (medium impact), D190 (smallimpact), H191 (small impact), and K194 (small impact). The bindingresidues of IL-7Rα to antibody HAL403b in descending order of mutanteffects were identified as follows: 182 (high impact on binding), K84(medium impact), K100 (medium impact), T105 (medium impact), Y192(medium impact), D190 (small impact), H191 (small impact), and K194(small impact).

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

It is claimed:
 1. An isolated interleukin-7 receptor (IL-7R) antibodywhich specifically binds to interleukin-7 receptor alpha (IL-7Rα) andcomprises an antigen binding region that cross-competes with amonoclonal antibody selected from the group consisting of P3A9, P4B3,P2D2, P2E11, HAL403a, HAL403b, C1GM and C2M3, for binding to IL-7Rα. 2.The antibody of claim 1, wherein the antibody binds to an epitopecomprising residues I82, K84, K100, T105, and Y192 of human IL-7Rα. 3.An isolated antibody which specifically binds to interleukin-7 receptoralpha (IL-7Rα), wherein the antibody comprises a heavy chain variableregion (VH) complementary determining region one (CDR1) having the aminoacid sequence X₁X₂VMH, wherein X₁ is D or N; X₂ is S or Y (SEQ ID NO:50), a VH CDR2 having the amino acid sequence X₁X₂X₃X₄X₅GX₆X₇TYYADSVKG,wherein X₁ is L or A; X₂ is V or I; X₃ is G or S; X₄ is W or G; X₅ is Dor S; X₆ is F, G or S; X₇ is F, A or S (SEQ ID NO: 51), and a VH CDR3having the amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈, wherein X₁ is Q or D;X₂ is G or I; X₃ is D or S; X₄ is Y or G; X₅ is M, V or G; X₆ is G or F;X₇ is N, D or M; X₈ is N, Y or D (SEQ ID NO: 52), a light chain variableregion (VL) CDR1 having the amino acid sequence TX₁SSGX₂IX₃SSYVQ whereinX₁ is R or G; X₂ is S or R; X₃ is D or A (SEQ ID NO: 53), a VL CDR2having the amino acid sequence EDX₁QRPS wherein X₁ is D or N (SEQ ID NO:54), and a VL CDR3 having the amino acid sequence X₁X₂YX₃X₄X₅X₆LX₇wherein X₁ is Q or M; X₂ is S or Q; X₃ is D or A; X₄ is F or S; X₅ is Hor S; X₆ is H or S; X₇ is V or W (SEQ ID NO: 55), wherein the antibodyblocks STAT5 phosphorylation in a STAT5 activation assay.
 4. An isolatedantibody which specifically binds to interleukin-7 receptor alpha(IL-7Rα), wherein the antibody comprises: a heavy chain variable region(VH) comprising the following complementarity determining regions(CDRs): a VH CDR1 that is a VH CDR1 in SEQ ID NO: 40; a VH CDR2 that isa VH CDR2 in SEQ ID NO: 40; and a VH CDR3 that is a VH CDR3 in SEQ IDNO: 40; and light chain variable region (VL) comprising the followingCDRs: a VL CDR1 that is a VL CDR1 in SEQ ID NO: 41; a VL CDR2 that is aVL CDR2 in SEQ ID NO: 41; and a VL CDR3 that is a VL CDR3 in SEQ ID NO:41.
 5. The isolated antibody of claim 4, wherein the VH region comprisesa VH CDR1 having the amino acid sequence DSVMH (SEQ ID NO: 19), GFTFDDS(SEQ ID NO: 46), or GFTFDDSVMH (SEQ ID NO: 47), a VH CDR2 having theamino acid sequence LVGWDGFFTYYADSVKG (SEQ ID NO: 23) or GWDGFF (SEQ IDNO: 48), and a VH CDR3 having the amino acid sequence QGDYMGNN (SEQ IDNO: 49).
 6. The isolated antibody of claim 5, wherein the VL regioncomprises a VL CDR1 having the amino acid sequence TRSSGSIDSSYVQ (SEQ IDNO: 29), a VL CDR2 having the amino acid sequence EDDQRPS (SEQ ID NO:31), and a VL CDR3 having the amino acid sequence QSYDFHHLV (SEQ ID NO:36).
 7. The antibody of claim 4, wherein the VH region comprises theamino acid sequence shown in SEQ ID NO: 40 and the VL region comprisesthe amino acid sequence shown in SEQ ID NO:
 41. 8. The antibody of claim7, wherein said antibody comprises a light chain having the amino acidsequence shown in SEQ ID NO: 43 and a heavy chain having the amino acidsequence shown in SEQ ID NO: 42, with or without the C-terminal lysineof SEQ ID NO:
 42. 9. The isolated antibody of claim 4, wherein each CDRis defined in accordance with the Kabat definition, the Chothiadefinition, the combination of the Kabat definition and the Chothiadefinition, the AbM definition, or the contact definition of CDR. 10.The antibody of any claim 4, wherein the antibody further comprises aconstant region.
 11. The antibody of claim 10, wherein the antibody isof the human IgG1 or IgG2Δa subclass.
 12. A pharmaceutical compositioncomprising the antibody of claim
 4. 13. A cell line that recombinantlyproduces the antibody of claim
 4. 14. A nucleic acid encoding theantibody of claim
 4. 15. A method for treating and/or preventing anautoimmune disorder in an individual, the method comprisingadministering a therapeutically effective amount of an antagonist IL-7Rantibody to an individual suffering from or at risk for an autoimmunedisorder, thereby ameliorating and/or preventing one or more symptoms ofthe autoimmune disorder, wherein the autoimmune disorder is selectedfrom the group consisting of type 1 diabetes, rheumatoid arthritis,lupus and multiple sclerosis.
 16. The method of claim 15, whereinadministration of the antagonist IL-7R antibody results in reduced naïveand activated T cell populations in the individual compared to beforeadministration.
 17. The method of claim 16, wherein the reduced T cellpopulations in the individual comprise T_(H)1 and/or T_(H)17 cells. 18.A method of treating and/or preventing type 2 diabetes in an individual,the method comprising administering a therapeutically effective amountof an IL-7R antagonist to an individual suffering from or at risk fortype 2 diabetes, thereby ameliorating and/or preventing one or moresymptoms of type 2 diabetes.
 19. The method of claim 18, wherein theIL-7R antagonist is an antagonist IL-7R antibody.
 20. A method fortreating and/or preventing graft-versus-host disease (GVHD) in anindividual, comprising administering a therapeutically effective amountof an antagonist IL-7R antibody to an individual suffering from GVHD,thereby ameliorating one or more symptoms of GVHD.